Isoprenoid synthases

ABSTRACT

The invention relates to the field of genetic engineering of flavor, fragrance and bio-control agent development. More specifically it relates to a process for production of natural flavors, fragrances or bio-control agents by the control of one or more genes implicated in that process. The invention provides an isolated or recombinant nucleic acid or functional fragment thereof encoding a proteinaceous molecule essentially capable of flavor, fragrance and/or bio-control agent synthesis when provided with a suitable substrate under appropriate reaction conditions. The invention further provides a nucleic acid or functional fragment thereof encoding a proteinaceous molecule essentially capable of synthesizing at least a monoterpene alcohol linalool when contacted with geranyl diphosphate (GPP) and/or at least a sesquiterpene alcohol nerolidol when contacted with farnesyl diphosphate (FPP) under appropriate reaction conditions.

This application is a International Application PCT/NL02/00089 filed on12 Feb. 2002, which designated the U.S., claims the benefit thereof andincorporates the same by reference.

The invention relates to the field of genetic engineering of flavor,fragrance or bio-control agent development. More specifically it relatesto a process for production of bioactive isoprenoid compounds by thecontrol or modulation of one or more genes implicated in that process.

Isoprenoids are the largest and most diverse group of plant secondarycompounds. At least 20,000 isoprenoids have been described and withoutdoubt many more will be discovered in the future. By definitionisoprenoids are made up of socalled isoprene (C5) units. This can berecognized in the number of C-atoms present in the isoprenoids whichusually can be divided by five (C5, C10, C15, C20, C25, C30 and C40),although also irregular isoprenoids and polyterpenes have been reported.Important members of the isoprenoids a.o. are the carotenoids,gibberellins, abscisic acid, some cytokinins, sterols, and theterpenoids, consisting of a.o. monoterpenes, sesquiterpenes, diterpenes,triterpenes, tetraterpenes and polyterpenes (rubbers), etc. Most ofthese compounds occur free but they can also be modified, or derivatizedas esters and glycosides, or attached to proteins. Among the isoprenoidsthere are many compounds with biological activity, for example as plantgrowth regulator (gibberellins, abscisic acid, cytokinins), and in theinteraction between plants and other organisms (for example asanti-microbials, infochemicals and as the isoprenoid germinationstimulants that are exuded by the roots of some plant species and inducethe germination of parasitic weed seeds).

Mono- and sesquiterpenes, the C10 and C15 branch of the isoprenoidfamily, were investigated for their economically interesting value asflavor and fragrance compounds in foods and cosmetics and theiranti-carcinogenic and antimicrobial properties. Mono- and sesquiterpeneshave also been shown to be of ecological significance, for instance inthe interaction between plants, plants and insects/spider mites andplants and microorganisms. Therefore, plants producing mono- andsesquiterpenes have been investigated by many authors and this hasresulted in a better understanding of the biochemical pathways leadingto the formation of these compounds and their derivatives.

Linalool is an acyclic monoterpene alcohol that has a peculiar creamyfloral, sweet taste. In Clarkia breweri (Onagraceae) linalool, amongstother compounds, is responsible for the attraction of pollinating moths.Linalool is one of the volatile compounds released as a semiochemicalafter herbivore attack in some plants and as such may attract predatorsof the herbivores. The sweet taste of linalool makes it suitable toenhance the blueberry flavor of foodstuffs especially of beverages (U.S.Pat. No. 4,041,185). Furthermore, linalool is known to have abroad-spectrum antimicrobial activity. It is reported by Pattnaik et al.(Microbios 89: 39-46, 1997) to display antibacterial activity againstGram-positive and Gram-negative bacteria as well as antifungal activityagainst yeast-like and filamentous fungi.

Nerolidol, the sesquiterpene analogue of the monoterpenoid linalool, isa component of many essential oils and flower headspaces (Bauer et al.,Common Fragrance and Flavor Materials. Preparations, Properties andUses, VCH Verlaggesellschaft, Weinheim, Germany, 1990; Knudsen et al.,Phytochemistry 33: 253-280, 1993). Nerolidol has been reported to haveanti-microbial activity. EP 0420630A2 describes the use of nerolidol inan antiplaque oral composition. Bouwmeester et al (Plant Physiol. 121:173-180, 1999) for cucumber and Lima bean and Degenhardt and Gershenzon(Planta 210: 815-822, 2000) for maize showed that nerolidol biosynthesisis induced upon respectively spider mite or Spodoptera feeding. Theenzyme responsible for the formation of nerolidol catalyses theregulatory step in the formation of the important signalling molecule4,8-dimethyl-1,3(E),7-nonatriene. Both nerolidol and4,8-dimethyl-1,3(E),7-nonatriene are important constituents of thevolatile blend produced in maize upon feeding of beet army worm larvae(Turlings et al., Science 250: 1251-1253, 1990; Degenhardt andGershenzon, 2000) and in gerbera in response to feeding of spider mites(Krips et al., J. Chem Ecol 1999). Also in the headspace of severalflowers, nerolidol is an important constituent often together with4,8-dimethyl-1,3(E),7-nonatriene (Kaiser, In: Perfumes: Art, Science andTechnology, Elsevier Science Publishers, Essex, UK, pp 213-250, 1991;Knudsen et al., 1993). Nerolidol has also been reported as a constituentof pheromone mixtures of a number of insects and spider mites (Aldrich-JR; Lusby-W R; Kochansky-J P, Experientia. 1986, 42: 5, 583-585; Regev-S;Cone-W W. Environmental-Entomology 1976, 5: 1, 133-138) and has beendescribed as being miticidal if formulated in a controlled releasesubstrate (U.S. Pat. No. 4,775,534).Also, nerolidol has been reported tobe an extremely effective repellent of mosquitoes.

From a number of plants, several cDNAs encoding enzymes involved in thebiosynthesis of monoterpenoids have been isolated such as S-linalool andR-linalool synthases (Cseke et al., Mol. Biol. Evol. 15: 1491-1498,1998; Jia et al., Arch Biochem Biophys 372: 143-149, 1999), (−)-4Slimonene synthase (Colby et al., J Biol Chem 268: 23016-23024, 1993;Bohlmann et al., J Biol Chem 272: 21784-21792, 1997).

WO 9715584 describes the use of S-linalool synthase, an acyclicmonoterpene synthase, in the genetic engineering of scent production.The use of the limonene (monoterpene) cyclase in the control of cornrootworm, by inserting a nucleotide sequence coding for limonene cyclaseinto the plants is described in WO 9637102. In WO 0022150 the use of alimonene synthase, linalool synthase and combination of limonene andcarveol synthase (actually called limonene hydroxylase) for the controlof insects is described. However, terpenoid products were only formed incombination with a GPP synthase.

The enzymes involved in the production of precursors for the synthesisof the primary monoterpene skeletons are all active in the plastids,since all cloned genes of this pathway until now have plastid targetingsignals. Recently, for one enzyme, (4S)-limonene synthase, localisationin the leucoplasts of the secretory cells in Mentha spicata has beendemonstrated with immunogold labeling. The plastid targeting signalsindicate that isoprenoid precursors for monoterpene metabolism areformed in the plastids, although some partitioning of these precursorsbetween the different cellular compartments in plants has been shown tooccur. Unlike other monoterpene (and diterpene) cyclases that bearcleavable transit peptides of 50-70 amino acids, the S-linalool synthasecDNA isolated by Pichersky and co-workers encodes a protein with anapparent cleavable peptide of maximally only eight amino acids long.Nevertheless, typical plastid targeting signal characteristics werefound in the first 60 amino acids of the cDNA, supporting that thelinalool synthase enzyme, as expected for a monoterpene synthase, isindeed targeted to the plastids. Two independent cDNA clones encoding5-epi-aristolochene synthase (EAS) from tobacco have been isolated andcharacterised by Facchini and Chappell (Proc Natl Acad. Sci. USA,89:11088-11092, 1992). Back and Chappell described the cloning andbacterial expression of vetispiradiene synthase found in Hyoscyamusmuticus (J. Biol. Chem., 270(13):7375-7381, 1995). The cDNA encodingamorpha-4,11-diene synthase, an intermediate in the biosynthesis of theanti-malarial artemisinin, was isolated and characterised by Mercke etal. (Arch. Biochem. Biophys., 381(1):173-180, 2000). Sesquiterpenebiosynthesis is compartmentalised to the cytosol, and none of the sofarisolated sesquiterpene synthases bear any targeting signal. Farnesyldiphosphate (FPP) is present in every living organism and it is theprecursor of a large number of primary and secondary metabolites. It hasbeen established that FPP is the precursor of all sesquiterpenoids.There are several thousands of different sesquiterpenoid compoundsidentified in many living organisms. Examples are the bittersesquiterpene lactones such as sonchuside A and C, and cichorilide A inchicory (De Kraker et al., Plant Physiol 117: 1381-1392, 1998). Thefirst committed step in the biosynthesis of these compounds is catalysedby a germacrene A synthase which was cloned from chicory (PCT/EP0002130). Other examples are the cloning of three sesquiterpenesynthases ((E)-α-bisabolene, δ-selinene, and γ-humulene synthase) fromgrand fir (WO 99/37139; Bohlmann et al., proc Natl Acad Sci, USA, 95:6756-6761), and a germacrene C synthase from tomato (Colby et al., ProcNatl Acad Sci, USA, 95: 2216-2221). The use of the amorpha-4,11-dienesynthase in the engineering of artemisinin biosynthesis is described inEP 0 982 404 A1. However, the putative sesquiterpene synthaseresponsible for the formation of the biologically important nerolidolhas never been cloned.

The use of recombinant DNA technology to introduce resistance based onsecondary metabolites in plants has had only limited success. Forexample, Hain et al (Nature 361, 153-156, 1993) succeeded in introducingfungal resistance in a number of plant species by the introduction ofthe resveratrol synthase cDNA, that they isolated from grape. Althoughthere are reports on the anti-microbial and insecticidal effects ofspecific terpenoids, resistance against fungi as a result of theexpression of a terpene synthase in plants has not been reported sofar.

The invention provides an isolated or recombinant nucleic acid orfunctional fragment thereof encoding a proteinaceous moleculeessentially capable of isoprenoid bioactive compound (herein alsoidentified as flavor, fragrance and/or bio-control agent) synthesis whenprovided with a suitable substrate under appropriate reactionconditions. Presently, the main way to produce plant flavor (for ease ofreference with flavor also fragrances are generally meant) compounds isby the synthetic route. Synthetic organic chemicals constitute more than80-90% (by weight and value) of the raw materials used in flavor andfragrance formulations. However, problems often exist concerningproduction. Extraction from intact plants and conventional fermentationare currently providing alternative routes for the commercial productionof flavor/aroma chemicals. However, the demand for natural flavors bythe consumer has been steadily increasing, and demand often outstripssupply. In many cases sought after flavor compounds can not easily beisolated. An understanding of the precursors and characterization ofgenes encoding enzymes involved in diverse pathways leading to theformation of flavors is essential for the production of natural flavors.The nucleic acids and their encoded proteinaceous molecules of thepresent invention are involved in the biosynthetic pathway for terpenoidproduction and as such they provide new means and methods for thein-vivo and in-vitro biotechnological production of bio-flavours,natural flavor chemicals and bio-control compounds.

In addition the nucleic acids and their encoded proteinaceous moleculesof the present invention and products synthesized are essentiallycapable acting as potent bio-control agents alone or in combination.

Fungi and bacteria have become an increasing threat to humans.Opportunistic microbial infections have increased dramatically in thelast two decades and have become a significant cause of morbidity andmortality. Over recent years, the frequency of life-threatening fungalinfections has increased dramatically, making fungal infections nowresponsible for nearly 40% of all deaths from hospital-acquiredinfections. Increased numbers of patients with an impaired immune system(such as due to ageing, severe burns, AIDS, chemotherapy against cancer,or immunosuppressive therapy for organ transplants), together with agrowing list of potential pathogenic fungi and bacteria are recognizedas factors contributing to this rising public health-hazard. There isonly a limited set of bio-control compounds available, and resistance toexisting bio-control drugs is becoming a problem of increasing concern.Also clinically used antimycotics may show harmful side effects.

Fungi are responsible for substantial economic losses due to foodspoilage caused by highly dangerous toxins (mycotoxins). To add to thisproblem food additives to prevent fungal contamination may also bepotentially carcinogenic. Additionally plant pathogenic micro-organismscause huge crop losses and this has promoted the extensive use ofpesticides all over the world. Some pesticides have deleterious effectson other organisms than the pests they are intended to control, on waterquality, and on the environment in general. Current antimicrobials areoften not specific enough, and several microbial species exhibitincreasing resistance to these pesticides. There is a need to developnew and more advanced bio-control agents with novel modes of action andbroad spectra directed against plant and animal pathogens. The nucleicacids and their encoded proteinaceous molecules of the present inventioninvolved in terpenoid biosynthesis, as such provide a new method for thein-vivo and in-vitro biotechnological production of natural and morespecific anti-microbials or bio-control agents, for example antifungals.

The nucleic acid as used herein refers to an oligonucleotide, nucleotideor polynucleotide, and fragments or portions thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represents the sense or antisense strand. A proteinaceous moleculeas used herein refers to a molecule comprising peptide or protein.Natural flavor synthesis as used herein refers to flavor and fragrancecompounds synthesized that are identical to their natural counterparts.Natural counterpart as used herein refers to products that are obtaineddirectly from plants and sometimes from animal sources by physicalprocedures. Synthetic flavors refers to nature identical compounds thatare produced synthetically but are chemically identical to their naturalcounterpart. Nature-identical compounds are with few exceptions the onlysynthetic compounds used in flavors in addition to natural products.Artificial flavor synthesis refers to flavor compounds that have notbeen identified in plant or animal products for human consumption. Thenucleic acids of the present invention pave the way for the productionof artificial flavors using techniques known in the art such as forexample combinatorial biosynthesis, metabolic pathway engineering, geneshuffling, directed evolution of proteins etc. Bio-control synthesis asused herein refers to bio-control compounds synthesized which can act asan bio-control agent. A bio-control agent as used herein refers to acompound, which can at least in part suppress or inhibit or restrict thegrowth of a pathogenic organism (e.g. fungi, bacteria etc.), that is acompound that has anti-pathogenic activity.

The invention further provides for a nucleic acid or functional fragmentthereof wherein said nucleic acid encodes a proteinaceous moleculeessentially capable of synthesizing at least a monoterpene alcohollinalool when contacted with geranyl diphosphate (GPP) and/or at least asesquiterpene alcohol nerolidol when contacted with farnesyl diphosphate(FPP) under appropriate reaction conditions. The definition ‘functionalfragment thereof’ means that a particular subject sequence may vary fromthe reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and the subject sequence.It may be advantageous to produce a nucleic acid according to theinvention or derivatives thereof possessing a substantially differentcodon usage. It is known by those skilled in the art that as a result ofdegeneracy of the genetic code, a multitude of gene sequences, somebearing minimal homology to the nucleotide sequences of any known andany naturally occurring genes may be produced. The invention includespossible variation of the nucleic acid sequence that could be made byselecting combinations based on possible codon choices. In additiondeliberate amino acid substitution may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity, and/or theamphipathetic nature of the residues as long as the biological activityof the polypeptide is retained.

In a preferred embodiment said nucleic acid encodes a terpene cyclasewhich has a combined nerolidol and linalool synthesizing capacity.Nerolidol is a sesquiterpene analog of the monoterpenoid linalool. Theenzymes involved in the production of precursors for the synthesis ofthe primary monoterpene skeletons have been shown to be active in theplastids. The ability of said terpene cyclase to synthesize linaloolappears to be influenced by the presence of a plastid targeting signalsequence that is rich in hydroxylated and basic residues. Sesquiterpenebiosynthesis is compartmentalised to the cytosol, and none of the sofarisolated sesquiterpene synthases bear any targeting signal. However, thepresent invention shows that monoterpenes can also be produced bycytosolic monoterpene synthases. Apparently the substrate GPP is presentin the cytosol. The invention shows that the production ofsesquiterpenes in the cytosol is hampered by a lack of substrate. Theco-expression of a cytosolic FPP-synthase or the transformation with afusion construct of sesquiterpene synthase and FPP synthase is nowprovided to overcome this problem. An additional solution is thetargeting of sesquiterpene biosynthesis to other cell compartments byadding or changing a targeting signal to/of the sesquiterpene synthaseand/or co-transformation of an FPP synthase with the same targeting ortransformation with a targeted fusion construct of sesquiterpenesynthase and FPP synthase. In addition to FPP synthase, other enzymescatalyzing committed steps in the biosynthesis of GPP and FPP throughthe mevalonate and non-mevalonate pathway can be coupled to orco-expressed with monoterpene and sesquiterpene synthases to increasethe levels of monoterpenes and/or sesquiterpenes produced. These enzymescan be directed (by adding, changing and removing targeting signals) todifferent compartments (i.e. mitochondria, chloroplasts, chromoplasts,leucoplasts, peroxisomes (see also example 7).

The invention thus provides a nucleic acid according to the inventionencoding a proteinaceous molecule provided with a targeting signal, suchas a plastid targeting or a mitochondrial targeting signal, or atargeting signal to any other organel or a nucleic acid according to theinvention encoding a proteinaceous molecule without such signal,depending on where synthesis is required. The invention thus provides anucleic acid according to the invention encoding a proteinaceousmolecule essentially capable of isoprenoid bio-active compound synthesisin the cytosol in a cell when provided with a suitable substrate underappropriate reaction conditions. Similarly, it provides a nucleic acidaccording to the invention encoding a proteinaceous molecule essentiallycapable of isoprenoid bio-active compound synthesis in a plastid in acell or in a mitochondrium in a cell when provided with a suitablesubstrate under appropriate reaction conditions.

In a preferred embodiment said nucleic acid as provided herein isprovided with a nucleic acid encoding a targeting signal and/or remnantsof a targeting signal. Preferably said targeting signal is a plastidtargeting signal. Said plastid targeting signal is preferably located inthe N terminus (N-terminal transit peptide) and may have a highabundance of serine residues and/or theronine and/or a low number ofacidic residues and/or rich in hydroxylated and basic residues. In onepreferred embodiment said targeting signal has a F (Phe), K (Lys), V(Val), F (Phe), N (Asn) motif and/or a D (asp) S (Ser), L (Leu), L(Leu), Xaa, S (Ser), S (Ser) motif, where Xaa is preferably P(pro) or S(Ser). In another, the target signal RRxxxxxxxxW is preferred. Inparticular the invention provides a nucleic acid encoding an essentiallysesquiterpene synthase bioactive fragment, said nucleic acid providedwith a targeting signal to provide the encoded gene product withmonoterpene synthase activity, or a nucleic acid encoding an essentiallymonoterpene synthase bioactive fragment, said nucleic acid deprived froman essentially plastid targeting signal to provide the encoded geneproduct with sesquiterpene synthase activity, and thus provides thevarious enzymes with a different activity as would be expected.

It is understood that through convergent or divergent evolution newproteins with altered functions may be created by this route. Themutations that lead to divergence are mostly single base substitutionsthat engender individual amino acid replacements, although other eventsleading to deletions or insertions also occur. The mutations may be in anucleic acid comprising the transit peptide and/or the open readingframe (ORF). The new protein usually contains many of the pre-existingfeatures. The original biological function may be restored by reversingmutations (e.g. single base substitutions) using techniques known in theart (e.g. site directed mutagenesis).

In a preferred embodiment through a single base substitution in apredecessor sequence of said nucleic acid sequence (e.g. H64NORL) theN-terminal transit peptide is restored. Restored as used herein meansthat a stop codon in the target signal is removed, for example through asingle base substitution, so that translation begins at the first ATG(Met) upstream of the target signal/transit sequence or target signalremnant. The predecessor sequence of said nucleic sequence is a sequence(common ancestor sequence) which has a stop codon in the target signalor the target signal remnant so that the translation of the proteinbegins at a second ATG (Met) truncating the target signal or the targetsignal remnant. It is conjectured that the presence of the restoredtarget signal or target signal remnant influences the synthesis oflinalool and/or nerolidol.

The invention provides for a nucleic acid according to the inventionwherein said proteinaceous molecule comprises a terpenesynthase/cyclase. Preferably said proteinaceous molecule comprises aterpene synthase (cyclase), the properties of which should resemblethose of other terpene synthases (cyclases). The invention furtherprovides a nucleic acid according to the invention wherein saidproteinaceous molecule comprises a sesquiterpenoid synthase/cyclase.Sesquiterpenoid synthases/cyclases participate in the biosynthesis ofmost sesquiterpenoids. Ionization of FPP to the farnesyl cation is thefirst step in the biosynthesis of a large number of sesquiterpenes. Theproducts of many of the sesquiterpenoid synthases/cyclases catalyzingthe formation of a terpenoid skeleton from the respective diphosphatesubstrates (FPP) are mostly cyclic hydrodrocarbons, with a fewexceptions such as for example the acyclic sesquiterpene alcoholnerolidol. None of the sofar isolated sesquiterpene synthases bear anytargeting signal.

The invention further comprises a nucleic acid according to theinvention wherein said proteinaceous molecule comprises a nerolidolsynthase/cyclase protein or functional fragment thereof. The nerolidolsynthase/cyclase protein is essentially capable of the synthesis of theacyclic sesquiterpene alcohol nerolidol.

The invention provides a nucleic acid wherein said nerolidolsynthase/cyclase comprises (3S)-(E)-nerolidol synthase. The inventionfurther comprises a nucleic acid according to the invention wherein saidsesquiterpene alcohol nerolidol comprises trans-nerolidol. The inventionfurther comprises a nucleic acid according to the invention wherein saidmonoterpene alcohol linalool comprises S-linalool.

The invention provides for a nucleic acid according to the inventionwherein said nucleic acid encodes a proteinaceous molecule comprising anamino acid sequence or functional fragment thereof that is at least 50%identical to H64MUT sequence, more preferred 53 or 60% homologous, andeven more preferred 70, 80, 90, 95 or 99% homologous to the sequence asshown in FIG. 2 or functional fragment thereof.

Homology is generally over the full-length of the relevant sequenceshown herein. As is well-understood, homology at the amino acid level isgenerally in terms of amino acid similarity or identity. Similarityallows for “conservative variation”, i. e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine. Deliberate amino acid substitution may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity, and/orthe amphipathetic nature of the residues as long as the biologicalactivity of the polypeptide is retained. In a preferred embodiment, allpercentage homologies referred to herein refer to percentage sequenceidentity, e.g. percent (%) amino acid sequence identity with respect toa particular reference sequence can be the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the reference sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, without considering any conservative substitutions as part ofthe sequence identity. Amino acid similarity or identity can bedetermined by genetic programs known in the art.

The invention further provides a nucleic acid or functional fragmentthereof according to the invention wherein said nucleic acid encodes aproteinaceous molecule essentially capable of the synthesis of at leastone monoterpenoid when contacted with geranyl diphosphate (GPP) underappropriate reaction conditions. The invention further provides anucleic acid according to the invention wherein said nucleic acidencodes a proteinaceous molecule essentially capable of the synthesis ofat least one monoterpenoid, wherein said monoterpenoid comprisesα-pinene and/or β-pinene (bicyclic terpene hydrocarbons) and/or sabineneand/or β-myrcene (acyclic monoterpene) and/or α-phellandrene and/orβ-phellandrene and/or α-terpinolene and/or α-terpineol and/orγ-terpinene. Preferably said proteinaceous molecule comprises a terpenesynthase (cyclase), the properties of which should resemble those ofother terpene synthases (cyclase). Even more preferred saidproteinaceous molecule comprises a monoterpenene synthase/cyclase.Preferably said monoterpenoid comprises an olefinic monoterpenoid.

The invention further comprises a nucleic acid according to theinvention wherein said nucleic acid encodes a proteinaceous moleculecomprising an amino acid sequence or functional fragment thereof that isat least 50% identical to SOSV sequence (see FIG. 6), more preferred 53or 60% homologous, and even more preferred 70, 80 or 90% homologous tothe sequence as shown in FIG. 6 or functional fragment thereof.Preferably said nucleic acid does not contain an insertion of twocytosine residues causing a frame-shift followed by a stop codon givingrise to a truncated open reading frame (ORF), as depicted in FIGS. 6 and7.

The invention further comprises a nucleic acid encoding a proteinaceousmolecule according to the invention obtainable from a eukaryote. Aeukaroyte as used herein comprises a cell or organism with amembrane-bound, structurally discrete nucleus and other well-developedsubcellular compartments. Eukaryotes as used herein include allorganisms except viruses, bacteria, and cyanobacteria (blue-greenalgae). Preferably said nucleic acid is obtainable from strawberryand/or maize and/or tea and/or cucumber and/or lima bean and/or cottonand/or thyme species and/or citrus species and/or eucalypt speciesand/or grapefruit and/or fungi and/or yeasts.

The invention further comprises a nucleic acid encoding a proteinaceousmolecule according to the invention obtainable from a prokaroyte. Aprokaryote as used herein comprises a cell or organism lacking amembrane-bound, structurally discrete nucleus and other subcellularcompartments e.g. bacteria, including archaebacteria and cyanobacteria(blue green algae).

The invention further comprises a nucleic acid encoding a proteinaceousmolecule according to the invention obtainable from invertebrateanimals. An arthropod is a member of a phylum of invertebrate animalsthat includes insects, arachnids (spiders and mites e.g. spider mites(Tetranychus urticae), aphids (e.g. Aphis gossypii, Myzus persicae), andthrips (Frankliniella occidentalis) and crustaceans (crabs, lobsters,pillbugs, shrimp, etc.).

In a preferred embodiment said nucleic acid encoding a proteinaceousmolecule according to the invention is obtainable from strawberry. Theinvention further provides a nucleic acid according to the inventionwherein said nucleic acid expression is repressed by auxin during fruitmaturation. Indole-3-acetic acid or auxin is a plant hormone that playskey roles in regulating cell division, extension, and differentiation.

The invention provides a proteinaceous molecule encoded by a nucleicacid according to the invention. The invention further provides a vectorcomprising a nucleic acid according to the invention. Preferably saidvector is a recombinant expression vector comprising a coding sequencewhich is operably linked to a promoter sequence capable of directingexpression of said coding sequence in a host cell for said vector, and atranscription termination sequence, in which the coding sequence is anucleic acid according to the invention. Preferably said nucleic acidhas been provided with means for nuclear targeting and/or integration ina host genome.

Methods which are well known in the art can be used to constructexpression vectors containing the nucleic acid of the invention, andappropriate transcriptional and translational controls. These methodsinclude in-vitro recombinant techniques. Exogenous transcriptionalelements and initation codons can be used and also can be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate to the cell system inuse. In the case of plant expression vectors, the expression of anucleic acid of the invention may be driven by a number of previouslydefined and yet to be defined promoters, including inducible anddevelopmentally regulated promoters. The invention further contemplatesthe use of the individual promoters of the nucleic acid of the presentinvention for this purpose. In particular any promoters particularlyresponsive to ripening events, wound-inducible or specific induciblepromoters (e.g. spider mite, insect etc. inducible promoters, which canbe isolated from plants that were fed upon by for example spider mitesor insects), may be used to drive the tissue specific expression of saidnucleic acid. In addition, viral promoters such as the 35S and the 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV. Promoters or enhancers derived from thegenomes of plant cells, tissue specific promoters i.e fruit specificpromoters, Fbp7 (Columbo et al. 1997; Plant Cell 9; 703-715), 2A11promoter (Pear et al., 1989, Plant Molecular Biology, 13:639-651), smallsubunit of Rubisco (Corruzzi et al., 1984; EMBO J 3:16; Broglie et al.,1984 Science 224:838-843) or timing specific promoters such as ripeningspecific promoters (the E8 promoter, Diekman and Fisher, 1988, EMBO J,7:3315-3320) may be used. Suitable terminator sequences include that ofthe Agrobacterium tumefaciens nopaline synthase gene (Nos 3′ end), thetobacco ribulose bisphosphate carboxylase small subunit terminationregion; and other 3′ regions known in the art. Methods known in the artcan be used to construct recombinant vectors which will express ‘sense’or ‘antisense’ nucleic acid. Antisense or partial sense or othertechniques may also be used to reduce the expression of said nucleicacid leading to the production of a flavour, fragrance and/orbio-control compound. Full length sense techniques may be used toincrease or reduce the expression of said nucleic acid leading to theproduction of a flavor and bio-control compound.

The invention further provides a replicative cloning vector comprising anucleic acid according to the invention and a replicon operative in ahost cell for said vector. The invention contemplates the use of yetnon-described biological and non biological based expression systems andnovel host(s) systems that can be can be utilized to contain and expressthe nucleic acid of the invention. The definition host cell as usedherein refers to a cell in which an foreign process is executed bybio-interaction, irrespective of the cell belongs to a unicellular,multicellular, a differentiated organism or to an artificial cell, cellculture or protoplast.

The invention further provides a host comprising a nucleic acidaccording to the invention or a vector according to the invention. Avariety of vector/host expression systems can be utilized to contain andexpress the nucleic acid of the invention. These include micro-organismssuch as bacteria (e.g. E coli, B subtilis, Streptomyces, Pseudomonads)transformed with recombinant bacteriophage, plasmid or cosmid DNAexpression systems, yeast (e.g S. cerevisiae, Kluyveromyces lactis,Pichia pastoris, Hansenula polymorpha, Schizosacch. Pombe, Yarrowia)transformed with yeast expression vectors; filamentous fungi(Aspergillus nidulans, Aspergillus orizae, Aspergillus niger)transformed with filamentous fungi expression vectors, insect cellsystems transfected with virus expression vectors (eg baculovirus,adenovirus, herpes or vaccinia viruses); plant cell systems transfectedwith virus expression vectors (e.g. cauliflower mosaic virus, CaMV,tobacco mosaic virus, TMV) or transformed with bacterial expressionvectors (e.g Ti or Pbr322 plasmid); or mammalian cell systems (chinesehamster ovary (CHO), baby hamster kidney (BHK), Hybridoma's, includingcell lines of mouse, monkey, human and the like. A host strain may bechosen for its ability to modulate the expression of the nucleic acid orto process the expressed proteinaceous molecule in the desired fashion.Such modifications of said proteinaceous molecule include acylation,carboxylation, glycosylation, phosphorylation and lipidation. Posttranslation processing which cleaves a ‘prepro’ form of saidproteinaceous molecue may also be important for correct insertion,folding and/or function. Different host cells which have the correctcellular machinery and characteristic mechanisms for suchpost-translational activities maybe chosen to ensure correctmodification and processing of the introduced, foreign proteinaceousmolecule.

The invention further provides a host comprising a nucleic acidaccording to the invention or a vector according to the inventionwherein said host comprises a prokaroytic cell. The invention furtherprovides a host comprising a nucleic acid according to the invention ora vector according to the invention wherein said host comprises aeukaryotic cell.

The invention further provides a host comprising a nucleic acidaccording to the invention or a vector according to the inventionwherein said host comprises a plant and propagating material thereof.The invention is particularly useful for enabling plants to producelinalool, nerolidol or a combination of the two. This enables breedingof plants with improved flavor/fragrance as described for linalool alonein WO 9715584, or improved resistance against micro-organisms or insectsas described in Examples 8, 9, 12, 13 and 14 and WO 0022150 for linaloolthat had however to be co-expressed with a GPP synthase.

The bacterial diseases to which resistance is provided herein includebut are not limited to:

Erwinia spp. (e.g. E. amylovora (fire blight) and E. carotovora),Clavibacter spp. (e.g. C. michiganense pv. Sepedonicum (bacterialringspot potato), Corynebacterium spp., Pseudomonas spp. (e.g. P.syringae pv. tomato), Xanthomonas spp. (X. campestris and X.vesicatoria), and Agrobacterium spp.

The fungal diseases to which resistance is provided herein include butare not limited to: Powdery mildew fungi (Sphaerotheca spp. (e.g. S.pannosa var. rosa. (rose), S. humuli (hop), S. fuliginea (cucurbits)),Podosphaera leucotricha (apple), Uncinula necator (grape), Erysiphespp.(e.g. E. cichoracearum (cucurbits, tomato), E. polygoni (beet)),Leveillula taurica (tomato), Microsphaera euonymi (squash)), Botrytisspp. (e.g. B. cinerea (grey mold)), Cladosporium spp. (e.g. C. fulvum(in tomato)), Sphaeropsis spp. (e.g. Sphaeropsis sapinea (tip blight ofpine), Cercospora spp. (C. beticola in beet, C. zeae-maydis in corn, C.sorghi in sorghum), Fusarium spp. (e.g. F. oxysporum f. niveum (wilt onwatermelon) F. graminearum and F. moniliforme (scab on wheat) F.moniliforme, F. oxysporum, F. subglutinans, F. proliferatum),anthracnose diseases (Apiognomonia veneta (in Sycamore, ash, oak, maple,and walnut), Colletotrichum trifolii (Alfalfa anthracnose),Colletotrichum coccodes (black dot in potato)), rust diseases (e.g.Puccinia recondita (leaf rust in wheat) and Uromyces appendiculatus(rust in bean)), Phytophtora spp. (P. infestans (late blight on potato),P. sojae (blight on soybean), P. megasperma f. sp. medicaginis (root rotin alfalfa)), spoilage fungi (Gibberella spp., Diplodia spp.,Penicillium, Aspergillus spp. Penicillium spp., Peacilomyces spp.),Verticillium spp. (e.g. V. albo-atrum (black root rot in strawberry),Septoria spp. (e.g. S. tritici and S. avenae f. sp. triticea (Septoriain wheat), S. lycopersici (Septoria leaf spot in tomato)), Sclerotiniaspp. (e.g. S. sclerotiorum (white mold of beans), Aphanomyces spp. (e.g.A. cochlioides (root rot in sugar beet), Alternaria spp. (e.g. A. solani(early blight in tomato), Magnaporthe spp. (e.g. M. grisea (blast inrice))

Insects

The insects to which resistance is provided herein include but are notlimited to Lepidoptera, Orthoptera, Homoptera, Hemiptera, especiallysquash bugs (Anasa tristis); green stink bug (Acrosternum hilare);Riptortus clavatus; Coleoptera, especially, Colorado potato beetle(Leptinotarsa decemlineata); three-lined potato beetle (Lematrilineata); asparagus beetle (Crioceris asparagi); Mexican bean beetle(Epilachna varivestis); red flour beetle (Tribolium castaneum); confusedflour beetle (Tribolium confusum); the flea beetles (Chaetocnema spp.,Haltica spp. and Epitrix spp.); corn rootworm (Diabrotica Spp.); cowpeaweevil (Callosobruchus maculatus); boll weevil (Anthonomus grandis);rice weevil (Sitophilus oryza); maize weevil (Sitophilus zeamais);granary weevil (Sitophilus granarius); Egyptian alfalfa weevil (Hyperapostica); bean weevil (Acanthoscelides obtectus); lesser grain borer(Rhyzopertha dominica); yellow meal worm (Tenebrio molitor);Thysanoptera, especially, western flower thrips (Frankliniellaoccidentalis); Diptera, especially, leafminer spp. (Liriomyza trifolii);plant parasitic nematodes especially the potato cyst nematodes(Globodera spp.), the beet cyst nematode (Heterodera schachtii) and rootknot nematodes (Meloidogyne spp.).

Resistance can also be conferred by the invention in a tritrofic manner.That is, the invention can be used to have (transgenic) plants producevolatiles such as linalool, nerolidol and dimethylnonatriene—which isderived in planta from nerolidol—constitutively upon feeding of insectsor spider mites. These volatiles are known, as shown in the presentinvention, to be attractive to predators of insects and spider mites andthese predators can efficiently exterminate the attacking herbivoresthus protecting the crop against its enemies.

Resistance can de determined by performing the appropriate test with theparticular organism but can be predicted as well by determining terpenecontent such as demonstrated in FIG. 30 and example 13 herein. Plant asused herein refers to eukaryotic, autotrophic organisms. They arecharacterized by direct usage of solar energy for their primarymetabolism, their permanent cell wall and in case of multicellularindividuals their open unlimited growth. In case of heterotrophicplants, the organisms are in an evolutionary context essentially derivedfrom autotrophic plants in their structure and metabolism. The inventionprovides a plant or a part, such as a stem, leave, tuber, root, fruit orseed or propagating material thereof transformed with the expressionvector according to the invention. The invention further provides aplant or part thereof which contains within its genome a vectoraccording the invention.

The invention provides a host comprising a nucleic acid according to theinvention or a vector according to the invention wherein said hostcomprises a plant cell. ‘Plant cell’ as used herein is anyself-propagating cell bounded by a semi permeable membrane andcontaining one or more plastids. Such a cell requires a cell wall iffurther propagation is required. Plant cell as used herein may be partof a whole plant or may be an isolated cell or part of a tissue whichmay be regenerated into a whole plant and includes for example, seeds,suspension cultures, embryos, meristematic regions, callous tissues,protoplasts, leaves, roots, shoots, gametophytes, sporophytes, pollenand microspores. The vector constructs according to the invention can beintroduced into plant cells by direct DNA transformation, or pathogenmediated transfection. The procedure or method for preparing atransformant can be performed according to the conventional techniqueused in the fields of molecular biology, biotechnology and geneticengineering. Manipulation of nucleic acid in plant cells may be carriedout using the Cre/lox site specific recombination system as outlined inpatent application WO9109957. The target plant may be selected from anymonocotyledonous or dicotyledonous plant species. Exemplary plantsinclude potato, tomato, petunia, maize, sorghum, cotton, soybean, beans,rape, alfalfa, asparagus, sweet potato and chrysanthemum. However, it isnot to be construed as limiting, in as much as microbes and insects mayinfest many other crops. Thus, the methods of the invention are readilyapplicable to numerous plant species, if they are found to besusceptible to the microbes or insect species listed hereinabove,including without limitation, species from the genera Medicago,Trifolium, Vigna, Citrus, Daucus, Arabidopsis, Brassica, Raphanus,Sinapis, Capsicum, Lycopersicon, Nicotiana, Solanum, Helianthus, Bromus,Asparagus, Panicum, Pennisetum, Cucumis, Glycine, Lolium, Triticum andZea.

The invention further provides a host comprising a nucleic acidaccording to the invention or a vector according to the inventionwherein said host comprises an insect cell. Insect cells such assilkworm cells or larvae themselves may be used as a host. For examplein one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign nucleic acid inSpodoptera frugiperda cells or in Trichoplusia larvae. The nucleic acidof the invention may be cloned into the nonessential region of thevirus, such as the polyhedrin gene, and placed under control of apolyhedrin promoter. Successful insertion of the nucleic acid willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S frugiperda cells or Trichoplusia larvae in which the nucleicacid is expressed [Smith et al. (1993) J Virol 46:584; Engelhard et al.(1994) Proc. Natl acad Sci, 91: 3224-7].

The invention further provides a host wherein said vector according tothe invention and said host expresses a nerolidol synthase/cyclaseprotein or polypeptide. Preferably said host exhibits suitableglycosyltransferase activity, whereby the produced linalool andnerolidol is converted and accumulated or stored in said host as itsrespective linaloylglycoside and nerolidylglycoside. Preferably saidhost contains the appropriate (inducible) glycosidase enzyme suitablefor the release of the respective linalool and nerolidol. Alternativelysaid host is provided with a nucleic acid coding for a suitable(inducible) glycosidase enzyme. Host which contain a nucleic acidencoding a proteinaceous molecule according to the invention may beidentified by a variety of procedures known in the art. These proceduresinclude, but are not limited to DNA-DNA, DNA-RNA hybridisation,amplification using probes (portions or fragments of said nucleic acid),protein bioassay or immunoassay techniques which include membrane,solution or chip based technologies for the detection and/orquantification of said nucleic acid and encoded proteinaceous molecule.

The invention further provides a host wherein said vector according tothe invention and said host expresses a monoterpenene synthase/cyclaseprotein or polypeptide.

The invention provides a method for producing a flavor, fragrance and/orbio-control compound comprising a) transforming or transfecting asuitable host with at least one nucleic acid encoding a proteinaceousmolecule according to the invention b) expressing said nucleic acid inthe presence of a suitable substrate c) optionally isolating the formedproduct. In a preferred embodiment said nucleic acid includes a restoredtarget signal or a target signal remnant, i.e. in those case whereplastid targeting is required. In a preferred embodiment of theinvention is a method to produce nerolidol and/or linalool and/orα-pinene and/or β-pinene (bicyclic terpene hydrocarbons) and/or sabineneand/or β-myrcene (acyclic monoterpene) and/or α-phellandrene and/orβ-phellandrene and/or α-terpinolene and/or α-terpineol and/orγ-terpinene or mixtures thereof by a) transforming/transfecting asuitable host b) expressing at least one nucleic acid of the inventionin the presence of a suitable substrate and c) isolating the formedproducts. In a preferred embodiment said host exhibits suitableglycosyltransferase activity, whereby the produced linalool and/ornerolidol is converted and accumulated or stored in said host as itsrespective linaloylglycoside and nerolidylglycoside. It is most easywhen said host already contains the appropriate (inducible) glycosidaseenzyme suitable for the release of the respective linalool andnerolidol. This is however not required, expression without saidglycosyltransferase and/or glycosidase activity is perfectly wellsuitable for most purposes and alternatively said host may even beprovided with a nucleic acid coding for a suitable glycosidase enzyme,when deemed required. For bio-control acitivity, it is even provided toexpress the compounds according to the invention without saidglycosyltransferase and/or glycosidase activity, and let the bio-controlactivity partly depend on said activity in the target organism, e.g.after uptake by an insect the insects saliva, or on the induction ofsaid activity after herbivory or fungal infection.

A method for producing a compound according to the invention is providedcomprising a) transforming or transfecting a suitable host with at leastone nucleic acid encoding a proteinaceous molecule according to theinvention b) expressing said nucleic acid in the presence of a suitablesubstrate c) optionally isolating the formed product, wherein said hostcomprises a microorganism, plant cell or plant. Micro-organism as usedherein refers to microscopic organisms such as for example Archaea,Bacteria, Cyanobacteria, Microalgae, Fungi, Yeast, Viruses, Protozoa,Rotifers, Nematodes, Micro-Crustaceans, Micro-Molluscs, Micro-Shellfish,Micro-insects etc.

The invention provides a method for producing a flavor, fragrance and orbio-control compound in a cell-free lysate expression system comprisingexpressing at least one nucleic acid encoding a proteinaceous moleculeaccording to the invention in the presence of a suitable substrate andoptionally isolating the formed product, wherein said free lysate systemcontains all the components necessary for expression and processing.Cell-free lysate expression system as used herein refer to cell-freetranslation/translocation systems known in the art, such as for examplerabbit reticulocyte lysate translation system.

The invention further provides a flavor and/or bio-control compoundobtainable by a method according to the invention. Preferably saidflavor and/or bio-control compound comprises at least a nerolidol and/orlinalool and/or α-pinene and/or β-pinene (bicyclic terpene hydrocarbons)and/or sabinene and/or β-myrcene (acyclic monoterpene) and/orα-phellandrene and/or β-phellandrene and/or α-terpinolene and/orα-terpineol and/or γ-terpinene or mixtures thereof.

The invention further provides use of a flavor compound according to theinvention in the processed food industry as an additive. Preferably as afood additive to enhance the flavor of syrups, ice-creams, ices, frozendesserts, yogurts, pastries, sauces, sweets, confectionery, baked goodsetc., and like products, for example the enhancement of blueberry flavor(U.S. Pat. No. 4,041,185). Strawberry is a popular fruit for naturalflavor ingredients because of its flavor, fragrance, aroma and scent.The invention provides the use of the nucleic acid according to theinvention, for the industrial production of ‘fruit’ flavors which arenatural to match the odor fidelity of the natural fruit. The inventionprovides for the production of novel flavors, fragrances and/orbio-control agents by the use of the nucleic acid according to theinvention, alone or in combination, to provide novel avenues forproduction. For example, the natural or the stereochemically purenerolidol may be used as a substrate for the semi-synthesis of flavorand fragrance compounds or insect repellents as described in U.S. Pat.No. 5,196,200A). The compounds of the present invention may be used toreplace potentially carcinogenic synthetic food additives currentlyused. The invention provides use of a flavor and/or bio-control compoundaccording to the invention as a disinfectant additive for example toobtain natural formulations and compositions such as antiplaque oralcompositions as described in EP 0420630). The invention further providesuse of a flavor and/or bio-control compound according to the inventionas a degreasing solvent and/or plasticiser and/or dye carrier.

The invention further provides use of a flavor and/or bio-controlcompound according to the invention as a flavoring and/or bio-controlagent for oral medications and vitamins. The invention further providesuse of a flavor compound according to the invention for providingadditional flavor/aroma in beverages, including alcoholic andnon-alcoholic beverages.

The invention further provides use of a flavor compound according to theinvention for enhancing or reducing plant flavor/aroma/fragrance/scent.

The invention further provides use of a flavor compound according to theinvention for enhancing the flavor/aroma of natural products and/orsynthetic products and/or artificial products. The invention furtherprovides use of a flavor compound according to the invention for theindustrial synthesis of nature identical flavor/aroma substances. In apreferred embodiment said flavor compound of the present invention isused for the production of novel combinations of artificial flavorsubstances.

The invention provides use of a flavor and/or bio-control compoundaccording to the invention as a pest control agent. Pest as used hereinis a general term for organisms (rats, insects, mites, micro-organismsetc.) which may cause illness or damage or consume food crops and othermaterials important to humans/animals. The nucleic acid of the presentinvention pave the way through plant breeding to produce crops at leastmore capable of controlling or even eliminating detrimental pestinfestations by enabling them to produce more terpenoid volatiles (plantvolatile allelochemicals) to repel the attacking pest and/or to attractnatural pest enemies to the crop. Preferably said terpenoid volatilescomprise nerolidol and/or linalool. The flavor and/or bio-controlcompounds of the present invention can be used as insecticides, insectrepellents, insect pheromones, miticides, scabicides, antimicrobialagents, anti-fungals, anti-herbivore feeding agents etc. For example,nerolidol has been reported to be an extremely effective repellent ofmosquitoes. Formulations containing natural nerolidol, producedaccording to the present invention, may therefore be used in mosquitocontrol.

In a preferred embodiment said compound according to the invention isused for control of the a) interaction between plants and insects b)interaction between plants and micro-organisms c) interaction betweenone plant and another.

The invention provides use of a flavor and/or bio-control compoundaccording to the invention as an anti-microbial agent. Anti-microbialagent as used herein refers to a compound which can at least in partsuppress or inhibit or restrict the growth of a pathogenic organism(e.g. fungi, bacteria, yeast etc.).

Preferably said compound may be used together with at least one othercompound having anti-microbial activity to augment or supplement saidanti-microbial activity (e.g. said compound can act synergistically withat least one other anti-microbial compound). The use of synergisticcombinations of anti-microbial agents has many advantages. One suchadvantage is that it minimizes the known risk associated with the use ofpotentially deleterious anti-microbial agents which can be used in lowerdosages to achieve the same effect. It also lowers risks associated withthe use of non specific/non-selective anti-microbial agents, for exampleas additives in food and non food products. Preferably said compound canbe used for crop treatment programs to reduce or eliminate the use ofharmful pesticides/biocides [e.g. spray treatments]. It can beincorporated into products as an bio-control agent [e.g. householdmaterials, detergents, food products etc.] or applied to products [e.g.as an external coating to leather products etc.] to reduce risk ofspoilage or contamination.

The invention further provides use of a flavor compound according to theinvention for providing flavor/aroma in cosmetics (inc. soap perfumes,perfume specialties and bases), creams, sun-protectant products, hairconditioners, cleaning products, personal care products, health careproducts (inc. all mammalian health care products). The inventionfurther provides use of a flavor compound according to the invention asa lengthening agent and fixative in perfumes or as a suspension aid foraluminium salts in anti-perspirants pharmaceuticals (e.g. deodorants).

The invention provides use of a nucleic acid according to the inventionas a molecular marker or diagnostic tool. Preferably as a molecularmarker for flavor formation [for example nerolidol and/or linalooland/or α-pinene and/or β-pinene (bicyclic terpene hydrocarbons) and/orsabinene and/or β-myrcene (acyclic monoterpene) and/or α-phellandreneand/or β-phellandrene and/or α-terpinolene and/or α-terpineol and/orγ-terpinene production] in plant breeding. Even more preferred as amolecular marker for fruit ripening (for example fruit ripening ofstrawberry and grapefruit). The nucleic acid according to the inventioncan be used as markers for the selection of crop species, such as forexample maize, cotton, apple, and cucumber, and any other cropsemploying a volatile release defense mechanism, with improved productionof volatile terpenoids (e.g. a predator attracting flavor (terpenoid)compound according to the invention) in response to feeding pests.

The invention further provides use of a flavor and/or bio-controlcompound according to the invention in the preparation of a composition.Suitable basis for compositions are known in the art. Preferably saidcomposition comprises at least nerolidol and/or linalool and/or α-pineneand/or β-pinene and/or sabinene and/or β-myrcene and/or α-phellandreneand/or β-phellandrene and/or α-terpinolene and/or α-terpineol and/orγ-terpinene, or mixtures thereof.

The invention further provides a composition comprising a flavor and/orbio-control compound according to the invention. Preferably saidcompositions are anti-fungal, miticidal, or pesticidal. For example amiticidal composition is usefel for controlling spider mite populations.Preferably said compositions comprise slow-release formulations whichcan be employed for fumigation purposes. For example fumigation inagriculture for the protection of crops against micro-organisms andpests e.g. insects, mites etc. Preferably said composition is in a formthat can be administered to a plant, animal (including human), food ornon-food product, industrial product etc.

The invention provides a composition comprising a flavor and/orbio-control compound according to the invention which is apharmaceutical. Suitable pharmaceutical compositions are known and theymay be in dosage forms such as tablets, pills, powders, suspensions,capsules, suppositories, injection preparations, ointments, eye dropsetc. The invention provides a composition comprising a flavor and/orbio-control compound according to the invention which is aneutraceutical.

The invention provides for use of a composition comprising a flavorand/or bio-control compound according to the invention for augmenting orenhancing the aroma and/or taste of food or non food products and/orprotection of food or non food products against fungal contaminationand/or pest infestation. For example chewing gums, medicinal products,detergents, cosmetics, confectionery etc. Preferably said compositionwill enhance the shelf life/preservation of food and non-food products(inc. industrial products).

The invention provides for use of a composition comprising a flavorand/or bio-control compound according to the invention for thebiological control of pests. For example administrating said compositionto a plant. Modes of administration can readily be determined byconventional protocols and may take the form of sprays, dissolublepellets etc.

The invention provides for use of a composition comprising a flavorand/or bio-control compound according to the invention for theprotection of stored products. For example for the protection of storedproducts against micro-organisms, insects and other pests. For examplethe protection of potatoes, flowerbulbs, onions etc. against Phytophtoraspp, Phoma spp, Fusarium, Botrytis spp and other stored productpathogens.

The invention provides for use of a composition comprising a flavorand/or bio-control compound according to the invention for theprevention or treatment of disease. For example for the treatment ofdental caries and/or dental plaque and/or skin disorders (dermatologicalformulations) and/or immunosuppressive, anti-leukaemia andanti-retroviral treatment. A preferred embodiment is that saidcomposition is suitable for human consumption or external application.

The invention provides for a method of treatment of disease comprisingadministering a composition according to the invention with a carrier toa suitable recipient. Preferably said carrier is a pharmaceuticallyacceptable carrier (e.g. drug carrier system) or inert carrier, such asa glycoside.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Headspace analysis using GC-MS of wild (A) and cultivatedstrawberry (B). Chromatograms are of m/z 93 (obtained in SCAN mode).Peaks: 1, α-pinene; 2, β-myrcene; 3, β-phellandrene; 4, γ-terpinene(tentative); 5, α-terpinolene (tentative); 6, α-terpineol; 7,carvylacetate; 8, linalool; 9, trans-nerolidol.

FIG. 2: Sequence alignment of H64 proteins H64NORS (SEQ ID NO: 19),H64MUT (SEQ ID NO: 21), H64VES (SEQ ID NO: 23). H64MUT nucleic acidsequence after the stop codon in H64NORL (SEQ ID NO: 17) (locationmarked) was changed into a Leucine residue. Black background correspondsto identical residues in all three sequences and gray backgroundcorresponds to identity between two out of the three sequences.

FIG. 3: targeting signals in the different H64 genes.

-   A. Protein sequence alignment of the different H64 fragments    obtained by PCR on genomic DNA and the same regions in cDNAs    isolated. Arrows indicate which one of the sequences do not have a    stop codon in this region. The RR motif is common in targeting    signals of monoterpene synthases. Black background corresponds to    identical residues in all seven sequences and gray background    corresponds to identity between at least three out of the seven    sequences. In FIG. 3A, the following sequences identifiers identify    the sequences as they are listed in the Sequence Listing (infra):    -   SEQ8C (H64NORU3/UP16) SEQ ID NO: 35    -   SEQ Q9C (H64NORU4/UP1) SEQ ID NO: 38    -   SEQ 4B (H64VES) SEQ ID NO: 23 (1-89)    -   SEQ Q6C (H64NORU1/W151) SEQ ID NO: 29 (1-81)    -   SEQ 7C (H64NORU2/UP3) SEQ ID NO: 32    -   SEQ3B (H64MUT) SEQ ID NO: 21(1-61)    -   SEQ5C (H64NORD1/W155) SEQ ID NO: 26 (1-61).-   B. Site directed mutagenesis and constructing H64MUT from H64NORL.    The 5′region of H64NORL and H64MUT is aligned between the two ATG    codons and the position of directed mutagenesis is marked by the    gray background. The stop codon T(U)GA in H64NORL was converted to a    codon encoding a leucine residue (CTA). In FIG. 3B, the following    sequences identifiers identify the sequences as they are listed in    the Sequence Listing (infra):    -   SEQ3A (H64MIUT5′) SEQ ID NO: 20 (145-247)    -   SEQ1A (H64NORL5′) SEQ ID NO: 16 (244-346).

FIG. 4: Expression of H64 genes analyzed by RNA gel blots and H64NORLcDNA as a probe.

-   A. Expression in vegetative (leaves) and reproductive (4 stages of    fruit development) tissues.-   B. Expression in ripe fruit of two wild cultivars (1, Plant Research    International line H1 and 2 Plant Research International line 92189)    and two cultivated cultivars (1, cultivar Calypso and 2, cultivar    Gorrella.-   C. Expression in fruits treated with or without the synthetic auxin    NAA. Strawberry fruit (cultivar Elsanta) at the white stage of    development were treated with lanolin paste containing 100 mM NAA.    Treated and control berries (paste with no NAA) were treated, left    on the vine for 7 days and then picked and used for RNA isolation.

FIG. 5: The pRSET B expression vector used for cloning and expression ofH64MUT/, SOSA and SOSV in E. Coli cells.

FIG. 6: Nucleic acid sequence alignment of the two cultivated SOSA cDNAscloned (MA and WS) and their homolog from the wild strawberry (SOSV).Black background corresponds to identical residues in all threesequences and gray background corresponds to identity between two out ofthe three sequences. The location of the CC insertion causing the frameshift and the stop codon following it is depicted. The stop codon at the3′ is the end of the ORF. In FIG. 6. the following sequences identifiersidentify the sequences as they are listed in the Sequence Listing(infra):

-   -   SEQ10A (SOSA/WS) SEQ ID NO: 39    -   SEQ11A (SOSA/MA SEQ ID NO:42    -   SEQ12A (SOSV) SEQ ID NO: 45.

FIG. 7: Alignment of the protein sequence of the different SOS cDNAsisolated. Black background corresponds to identical residues in all fivesequences and gray background corresponds to identity between at leastthree out of the five sequences. The insertion of CC in SEQ11B(SOSA/MA)and SEQ10B(SOSA/WS) forms a proline residue and a stop codon after. InSEQ11C(SOSA/MA) and SEQ10C(SOSA/WS) the two cytosine nucleotides wereremoved and allowed further translation of the protein. In FIG. 7, thefollowing sequences identifiers identify the sequences as they arelisted in the Sequence Listing (infra):

-   -   SEQ11B (SOSA/MA) SEQ ID NO:43    -   SEQ 12B (SOSV) SEQ ID NO: 46    -   SEQ10B (SOSA/WS) SEQ ID NO: 40    -   SEQ10C (SOSA/WS) SEQ ID NO: 41    -   SEQ11C (SOSA/MA) SEQ ID NO:44.

FIG. 8: Nucleic acid sequence alignment of the different SOS fragmentsobtained by PCR on genomic DNA and the same regions in cDNAs isolatedfrom wild and cultivated strawberry. The source of the fragment ismarked in the left side of each sequence name. Black backgroundcorresponds to identical residues in all fifteen sequences and graybackground corresponds to identity between at least twelve out of thefifteen sequences. In FIG. 8, the following sequences identifiersidentify the sequences as they are listed in the Sequence Listing(infra):

-   DNA_SEQ23B (SOSA5/W74) SEQ ID NO:75    -   DNA_SEQ23B (SOSA6/256) SEQ ID NO:78    -   DNA_SEQ24B (SOSA7/W61) SEQ ID NO:81    -   DNA_SEQ21B (SOSA4/W59) SEQ ID NO:72    -   DNA_SEQ19B (SOSA2/W68) SEQ ID NO:66    -   DNA_SEQ15B (SOSV3/W90) SEQ ID NO:54    -   DNA_SEQ17B (SOSV5/W84) SEQ ID NO:60    -   DNA_SEQ20B (SOSA3/W46) SEG ID NO:69    -   DNA_SEQ13B (SOSV1/W76) SEQ ID NO:48    -   DNA_SEQ16B (SOSV4/W79) SEQ ID NO:57    -   DNA_SEQ18B (SOSA1/W66) SEQ ID NO:63    -   DNA_SEQ10A (SOSA/WS) SEQ ID NO:39 (629-833)    -   DNA_SEQ11A(SOSA/MA) SEQ ID NO:42 (1322-1526)    -   DNA_SEQ14B (SOSV2/W93) SEQ ID NO:51    -   DNA_SEQ12A (SOSV) SEQ ID NO:45 (635-839).

FIG. 9: Expression of SOS genes analyzed by RNA gel blots and SOSV cDNAas a probe

Expression in ripe fruit of two wild cultivars (1, Plant ResearchInternational line H1 and 2 Plant Research International line 92189) andtwo cultivated cultivars (1, cultivar Calypso and 2, cultivar Gorrella.

FIG. 10: Radio-GLC analysis of radio-labelled products formed from[³H]-geranyl diphosphate in assays with recombinant proteins. A, FIDsignal showing unlabelled authentic standards of 1, β-myrcene; 2,trans-ocimene; 3, linalool; 4, α-terpineol; 5, nerol; 6, geraniol. B,C,radio-traces showing enzymatic products of recombinant proteins SOSV (B)and H64MUT (C).

FIG. 11: Radio-GLC analysis of radio-labelled products formed from[³H]-farnesyl diphosphate in assays with recombinant protein. A, FIDsignal showing unlabelled authentic standards of 7, cis-nerolidol; 8,trans-nerolidol; 9, trans-trans-farnesol. B, radio-trace showingenzymatic products of recombinant protein H64MUT.

FIG. 12: GC-MS analysis on an HP5-MS column of products formed fromgeranyl diphosphate in assays with recombinant SOSV protein. Peaks: 1,α-pinene; 2, β-pinene; 3, sabinene; 4, β-myrcene; 5, α-phellandrene; 6,β-phellandrene; 7, dihydromyrcenol (tentative); 8, α-terpinolene(tentative); 9, α-terpineol (tentative).

FIG. 13: GC-MS analysis on an HP5-MS column of the product formed fromgeranyl diphosphate in an assay with recombinant H64MUT protein. A, m/z93 chromatogram. B, mass spectrum of the major product peak (linalool).

FIG. 14: GC-MS analysis on an HP5-MS column of the product formed fromfarnesyl diphosphate in an assay with recombinant H64MUT protein. A, m/z93 chromatogram. B, mass spectrum of the major product peak (nerolidol).

FIG. 15: Transient GFP expression of fusion proteins in tobaccoprotoplasts. g, GFP; ca, chlorophyll auto-fluorescence; mt, MitoTracker(mitochondrial stain); ol, overlay of chlorophyll auto-fluorescenceimage and GFP image; ol-mt, overlay of chlorophyll auto-fluorescenceimage, GFP image and Mitotracker image. 10 different constructs weremade (C1-C10) to study fragments derived from H64NORL (C1, C2), H64TAR4(C3, C4, C5) and H64VES (C7, C8, C9). See FIG. 16 for a schematicrepresentation of the different constructs made and used for thelocalization studies. C6 shows localization of fusion of a citruslimonene synthase 5′ end with GFP. C10 is a fusion of the H64VES regionbetween the two Methionine residues and the region down stream of thesecond Methionine from H64NORL. pOL65 is the original vector, containingonly GFP and was used to insert all fragments for fusion with the GFP.Rpo-ol is a positive control for plastidic targeting signal.Chloroplasts are on average 5 micrometer in size while mitochondria are1 micrometer in size. pOL65, C1, C2, C4, C5, C8 and C9 all showcytosolic localization. C3 shows dual plastidic and mitochondriallocalization. C6, C7, C10 and Rpo-ol show plastidic sub-cellularlocalization.

FIG. 16: Schematic representation of the different constructs used forGFP transient expression assays in tobacco protoplasts. Fagments derivedfrom the 5′-end of the cDNAs described in the invention were used for atranslational fusion with the GFP gene. The MID motif is present in mostsesquiterpene synthase genes described up to date. SC, stop codon. M1and M2 are the two methionine residues at the N-termini of the variousproteins (see also FIG. 3A).

FIG. 17: Comparison of effects of farnesol and linalool present in thegrowth medium on mycelium growth of Phytophthora infestans.

FIG. 18: Dose-response data of effects of linalool present in the growthmedium or the vapour phase on mycelium growth of Phytophthora infestans.

FIG. 19: Dose-response data of effects of nerolidol present in thegrowth medium or the vapour phase on mycelium growth of Phytophthorainfestans.

FIG. 20: Dose-response data of effects of linalool and nerolidol presentin the growth medium alone and in combination on mycelium growth ofPhytophthora infestans.

FIG. 21: Dose-response data of effects of linalool and nerolidol presentin the growth medium alone and in combination on mycelium growth ofPhytophthora infestans.

FIG. 22

Dose-response data of effects of linalool and nerolidol present in thegrowth medium on mycelium growth of Fusarium spp. on day 7.

FIG. 23

Dose-response data of effects of linalool and nerolidol present in thegrowth medium on mycelium growth of Botrytis spp. on day 7.

FIG. 24

Dose-response data of effects of nerolidol (A) and linalool (B) presentin the growth medium on spore germination of Fusarium verticillioidesisolates on day 3.

FIG. 25

Headspace analysis of transgenic Arabidopsis expressing the H64NORS withthe H64VES targeting signal (H64TAR) cDNA, showing a large peak oflinalool (1), and a smaller peak of nerolidol (2). Both compounds areabsent in control, wildtype Arabidopsis (see insert).

FIG. 26

Headspace analysis of volatiles produced by control and transgenic,H64TAR expressing, potato (3 individual transformants TM 9, TM 13, TM29). Linalool is virtually absent in control potato, and stronglyenhanced in the transgenic lines. Also 8-hydroxylinalool is enhanced inthe transgenic lines.

FIG. 27

Chiral analysis of the free linalool in control and transgenic potato,showing the presence of both enantiomers in control potato (about80:20). In the H64TAR trangenic lines the ratio has shifteddrammatically to the S-enantiomer, that is produced by the introducedenzyme.

FIG. 28A

Identification of linalyl-β-D-glucopyranoside in Petunia tissue usingHPLC-MS/MS. Ion trace m/z 375 of A: the synthesized(R,S)-linalyl-β-D-glucopyranoside, B: the transgenic Petunia leaf tissueand C: The control Petunia leaf tissue.

FIG. 28B

Product ion spectrum of A: The synthesized(R,S)-linalyl-β-D-glucopyranoside and B: The compound isolated from thetransgenic Petunia tissue. Retention time and product ion spectrum ofthe synthesized (R,S)-linalyl-β-D-glucopyranoside fit with the compounddetected in the transgenic Petunia tissue.

FIG. 29

Determination of the enantiomeric distribution of S- and R-linaloolafter enzymatic hydrolysis of the glucoside fraction obtained from leaftissue using chiral phase MDGC-MS analysis, A: Control tissue and B: thetransgenic tissue. The transgenic plant accumulates highly enrichedS-linalyl-β-D-glucopyranoside.

FIG. 30

FIG. 30 combines the data of table 1 and 4. FIGS. 30 A, B and C providethe correlation in lesion size, lesion growth rate, and sporulationrespectively of Phytophthora infestans isolate IPO 428-2 plotted againstthe content of linalool, 8-hydroxylinalool, linalooltriol,lynalylglucoside, 8-hydroxylinalylglucoside and linalyltriolglucosidecontent of the potato transgenic lines T or TM-9, -13, -29 and a controlline. The control data from table 4 on fungal growth and sporulationwere taken to be the average values of the H64NOR plants with negligibleincreased levels of either linalool, nerolidol or derivatives. Thelinalool (derivative) data provided in table 1 are much more reliableand quantitative than the SPME data on linalool in table 4, whichjustifies their use. FIG. 30 D provides the in vitro data on thesensitivity of Phytophthora infestans isolate IP0428-2 which was usedfor the in planta experiments to pure linalool in the medium asdescribed in Example 9.

FIG. 31

GC-MS spectra of transgenic and wildtype Arabidopsis plants. Peak 22.52was identified to be (E)-nerolidol, peak 13.74 was identified to bedimethylnonatriene.

EXAMPLES

The following examples are offered by way of illustration.

Example 1 Analysis of Terpenes in Wild and Cultivated Strawberry

Terpenoid Biosynthesis in Wild and Cultivated Strawberries

The cultivated variety (Elsanta) used by us for the mentionedexperiments produces both the monoterpene linalool and the sesquiterpenenerolidol. On the other hand the wild cultivar used (PRI line 92189)produces low levels of linalool but does not show a trace of nerolidol.Both literature reports and our own GC-MS data show similar patterns oflinalool and nerolidol production in several other cultivated and wildstrawberry varieties. Our sequencing data and experiments using therecombinant enzymes produced in E. coli show that the capability of thecultivated variety to form nerolidol was acquired by removing (bydeletions and translation stop) the targeting signal to the plastid[were the substrate for monoterpene biosynthesis is available (GPP)] andby directing the translation start to the downstream AUG codon. However,linalool in the cultivated varieties may also be formed by enzymesencoded by genes similar to H64TAR2, H64TAR4 and H64TAR6 which contain aproper targeting signal with no stop and therefore their proteinproducts are directed to the plastid for forming linalool. If GPP ispresent in the cytosol, then linalool could also be produced there by anenzyme encoded by a cytosolically expressed cDNA. We can not excludethat translation in H64TAR2, H64TAR4 and H64TAR6 may also start from thedownstream AUG codon (the one downstream from the RR motif and not theadditional AUG codon present just prior the RR motif) and this willresult in the formation of nerolidol as well. However, since cultivatedvarieties like the ones used in this study are mostly octaploids it islikely that evolutionary processes as polyploidity allows the plant toform an additional (mutated) gene from an existing gene and to producean additional beneficial compound such as nerolidol for flavour anddefense. Williams et al., (Biochemistry 1998,37,12213-12220) described arole for the tandem arginines present in the N-terminal of monoterpenesynthases in the unique diphosphate migration step accompanyingformation of the intermediate 3-s-linalyl diphosphate and preceding thefinal cyclization reaction catalyses by the monoterpene synthases. ThisRR motif is present in H64TAR2, H64TAR6, and H64VES and this mightexplain the formation of linalool by this genes encoding enzymes.However, the H64MUT recombinant protein does not contain the RR motifbut catalyses the formation of both nerolidol and linalool. This mightimplicate other residues between the RR motif location and the downstream AUG as functioning to determine whether monoterpene will beformed. This motif contain 12 amino acids: N-termini-DSLLPSSITIKP (SEQID NO:1).

The short genomic DNA sequence obtained (H64W149) contains an RW motifinstead of an RR motif and it might be of importance for the formationof the monoterpene linalool. In the wild cultivars (diploid) only onevariant encoding a protein with a targeting signal could be identified(both by PCR on either DNA and RNA) which may only catalyze theformation of the low levels of linalool detected.

Headspace analysis. Samples of ripe or ripening fruits were enclosed in1-L glass jars that were closed with a teflon-lined lid equipped with anin- and outlet, and placed in a climate room at 25° C. and 210μmol·m⁻²·s⁻¹ provided by 400-W HPI-T lights (Philips, Eindhoven, theNetherlands). A vacuum pump was used to draw of air through the glassjars at approximately 100 mL min⁻¹, with the incoming air being purifiedthrough a glass cartridge (140×4 mm) containing 150 mg Tenax TA (20/35mesh, Alltech, Breda, the Netherlands). At the outlet the volatilesemitted by the fruits were trapped on a similar Tenax cartridge.Volatiles were sampled during 24 h. Cartridges were eluted using 3×1 mLof redistilled pentane-diethyl ether (4:1). Of the (non-concentrated)samples, 2 μL were analysed by GC-MS using an HP 5890 series II gaschromatograph equipped with an HP-5MS column (30 m×0.25 mm i.d., 0.25 μmdf) and an HP 5972A Mass Selective Detector. The GC was programmed at aninitial temperature of 45° C. for 1 min, with a ramp of 10° min⁻¹ to280° C. and final time of 5 min. The injection port (splitless mode),interface and MS source temperatures were 250, 290 and 180° C.,respectively, and the He inlet pressure was controlled by electronicpressure control to achieve a constant column flow of 1.0 mL min⁻¹.Ionization potential was set at 70 eV, and scanning was performed from48-250 amu.

The analysis of the headspace profiles was focused on terpenoids by onlyshowing the ion 93 chromatogram (although samples were analysed usingthe SCAN mode). In that way, remarkable differences can be seen betweencultivated and wild strawberry: the headspace profile of the wildstrawberry contains carvylacetate and a number of olefinic monoterpenessuch as α-pinene, myrcene, α-phellandrene, and α-terpinolene,α-terpineol and γ-terpinene (the last three tentatively identified)(FIG. 1A), whereas the cultivated is dominated by two major peaks only:linalool and transnerolidol (FIG. 1B).

Example 2 General Molecular Techniques

DNA was isolated from young strawberry leaves as described by Marty etal., [Theor. Appl. Genet. (2000) 100:1129-1136].

RNA gel blots experiments were performed as described by Aharoni et al.,[The Plant Cell, (2000) 12, 647-661].

Cloning full length cDNAs was performed by using the SMART RACE cDNAAmplification Kit (Clontech) according to the manufacturer instructionswith slight modifications either to annealing temperatures (normallyreduced by 5 to 10° C. compared to the one recommended) or amount ofcycles (up to 35 cycles). PCR, restriction digests, plasmid DNAisolation and gel electrophoresis were performed using standardprotocols. All fragments were purified out of gel using the GFXpurification kit (Amersham). Cloning of PCR fragments was either done tothe PCR SCRIPT (Stratagene) or pCR 4Blunt-TOPO (Invitrogen) vectors (forblunt end products generated when using pfu polymerase) or to the PGEM-TEasy (Promega) vector (when A tailed PCR products were generated by theuse of taq polymerase). Throughout the text the following construct/cDNAnames will be used (also see sequence listing):

-   -   H64VES: wild strawberry, full length cDNA (with targeting        signal)    -   H64NORL: original cultivated strawberry cDNA starting from Met        1, including stopcodon between Met 1 and Met 2 (non-functional        targeting signal)    -   H64NORS: derived from H64NORL starting from Met 2 (no targeting        signal)    -   H64MUT: derived from H64NORL; stopcodon repaired    -   H64TAR: used for transformation of plants: composed of H64VES        Met1 to Met 2 region and H64NORS (with targeting signal)    -   H64NOR: used for transformation of plants: H64NORS including        intron

Example 3 Construction of a Strawberry Red Fruit Stage cDNA Library,Mass Excision and Random Sequencing

Messenger RNA Isolation and cDNA Library Construction

Total RNA was isolated from strawberry fruit red stage of developmentusing the method described by Manning K. [Analytical Biochemistry (1991)195, 45-50]. The cultivar used was Fragaria×ananassa Duch. cv. Elsanta.The cDNA library was produced as a custom service by (Stratagene) in thelambda zap vector. Messenger RNA was isolated from total RNA using thepolyA+ isolation kit (Pharmacia).

Mass Excision and Random Sequencing

The ExAssist™/SOLR™ system (Stratagene) was used for mass excision ofthe pBluescript SK(−) phagemid. The excision was done according to themanufacturer instructions using 20×10³ pfu from the non-amplifiedlibrary for each excision. High quality plasmid DNA from randomly pickedcolonies was extracted using the QIAGEN BioROBOT 9600. Colonies weregrown overnight in 3 ml Luria Broth medium (10 g/l tryptone, 5 g/l yeastextract, 5 g/l NaCl) supplemented with 100 mg/l ampicillin, centrifugedat 3000 RPM for 10 min. and the pellet was used directly for plasmid DNAisolation by the robot. Each DNA isolation round consisted of 96cultures.

Insert size was estimated by agarose gel electrophoresis afterrestriction enzyme digestion of the pBlueScript (SK-) vector with EcoRIand XhoI. Inserts with length above 500 bp were used for sequencing.Plasmid DNA from the selected samples were used for polymerase chainreaction (PCR) sequencing reactions using the ABI PRISM™ Dye TerminatorCycle Sequencing Ready Reaction Kit and the MJ Research PTC-200 DNAEngine™ thermal cycler. The T3 and T7 universal primers were used forsequencing from the 5′ and 3′ends respectively. PCR program wasaccording to the Dye Terminator manufacture's protocol (ABI PRISM). TheABI 373, 370A and 310 sequencers (Applied Bio-systems) were used.Sequences were edited manually to remove vector and non reliablesequences and submitted to the BLAST homology search (Altschul et al. J.Mol. Biol. 215, 403-410, 1990) provided by the National Center forBiotechnological Information on the world wide web(info@ncbi.nlm.nih.gov). Search was performed against all non-redundantdata bases available by the program.

Example 4 Cloning and Characterization of H64 Genes from Wild andCultivated Strawberry

Cloning of the H64 cDNA from Cultivated Strawberry (H64NORL) and itsHomologue from the Wild Strawberry (H64VES)

We primarily identified the H64 cDNA out of our randomly sequencedclones originating from the cultivated strawberry cultivar Elsanta (ripered fruit) cDNA library. Homology search results using the BLAST programindicated that the cDNA might encode a terpene synthase protein. Theentire H64 cDNA is 1874 bp long [(termed H64 Normal Long (H64NORL)] andcontains a open reading frame (ORF) encoding a 519 amino acids (aa) longprotein [we termed the part of the cDNA forming the 519 aa ORF as H64Normal Short (H64NORS), see FIG. 2].

Cloning of the wild strawberry homolog of the cultivated H64 cDNA wasaccomplished by the use of the SMART RACE kit (Clontech) using RNA fromthe Plant Research International collection of wild strawberries (line92189). Oligonucleotides primarily used for sequencing the H64NORL cDNAwere used for 3′ RACE amplification (AAP291-5′-CTTCATGAGGTTGCACTTCG-3′(SEQ ID NO: 2) and the nested oligonucleotide AAP293-5′-AATGGTGGAAGGAGCTTGGATTGG-3′ (SEQ ID NO: 3)). The full length wildstrawberry cDNA [H64 Vesca (H64VES)] was obtained by designing anoligonucleotide on the 3′ untranslated region (UTR) based on the 1000 bpfragment obtained in the 3′ RACE and using it to RACE for the 5′ side(5′ GTTCAACTCCACTTCCAGCAGTC 3′(SEQ ID NO: 4)). The H64VES cDNA is 1894bp long and contains a open reading frame encoding a 580 amino acids(aa) long protein. Sixty one amino acids downstream of the firstmethionine residue of the 580 aa protein we could identify an additionalmethionine residue. This 61 amino acids resemble the characteristicplastidic targeting signal of monoterpene synthases since it containsthe two arginines motif and a large number of serine residues [Williamset al. (Biochemistry, 37 12213-12220, 1998); see FIG. 3A). The H64NORLand H64VES cDNAs share 96% identity at the nucleic acid level and if thestop codon is eliminated and the rest of the sequence translated, 92.4%at the amino acid level (from the ATG located at nucleotide 145 up tothe end of the coding region). H64VES and H64NORS share 97.2% identityat the nucleic acid level and 94.2% at the amino acid level (when thepart starting from the beginning of H64NORS from H64NORVES is used forthe alignment up to the end of the coding region).

Analysis of H64 Expression During Development, in Cultivated and WildCultivars and in Response to Auxin Treatment

RNA gel blot analysis using H64NORL as a probe revealed that it isupregulated during the cultivated strawberry fruit ripening (FIG. 4). Noexpression could be detected in the leaf and green fruit tissues. H64expression increased from the white to red stage of fruit development.Analysis of H64 expression in ripe fruits of two wild and two cultivatedcultivars showed that H64 is strongly expressed in the cultivatedcultivars and hardly any expression could be detected in the wildcultivars (slight signal was detected in the wild cultivars after longexposure of the film, data not shown). Another RNA gel blot showed thatH64 is repressed by auxin. This correlates with the fact that also otherripening up-regulated genes in strawberry are repressed by auxin.

Site Directed Mutagenesis of H64NORL

A more thorough analysis of the H64 cDNA (termed H64NORL) revealed thatit might contain an additional ATG start codon, 99 bp upstream of theoriginal ATG we identified (proposed to be the beginning of the ORFencoding the 519 aa H64NORS protein). The two ATG codons were located inframe but no peptide could be formed between them since a stop codonlocated 39 bp before the down stream ATG was evident. We suspected thatthe part between the two ATG is actually part of the protein and forsome reason it might be mutated so a shorter protein starting for thedownstream ATG might be formed. Additional support to this idea was thehigh abundance of serine residues identified in the translated areabetween the two ATGs. It resembled N-termini of other monoterpenoidsynthases which contain relatively high abundance of serine residues. Wetherefore employed site directed mutagenesis in order to modify the stopcodon and construct a non truncated H64NORL protein [termed H64Mutagenized (H64MUT)]. By changing the stop codon (TGA) into a leucineresidue (CTA) the H64MUT cDNA is 1659 bp long containing a 552 aa longprotein (see FIG. 3B). The site directed mutagenesis was performed usingthe QuikChange kit as described by the manufacturer (Stratagene). Theoligonucleotide used for the exchange was,5′-GGGAAGCAAGCTATCTAGAAAGTAGCAG-GCAATT-3′ (SEQ ID NO: 5).

PCR on Cultivated Strawberry Genomic DNA

In order to verify whether the sequence we obtained for H64NORL was nota PCR artifact and the stop codon between the two ATGs exists, weperformed PCR on the cultivated strawberry genomic DNA. We designed twooligonucleotides one upstream the first ATG (5′-CTCCCACAGCTTCTTAGTTGC-3′(SEQ ID NO: 6)) and the other downstream of the second ATG (thebeginning of H64NORS) (5′-CTAGCTCTGCTACATTCCTCAAGAC-3′ SEQ ID NO: 7)).Amplification with these two oligonucleotides was expected to amplify afragment of approximately 200 bp. We obtained two clear fragments of 300bp and 400 bp each. Sequencing four clones of the 300 bp lengthfragments revealed them to be similar to the original H64NORL cDNA.Sequencing and aligning 20 of the larger clones identified severalisoforms which were different from the original cultivated H64NORL cDNA.All fragments (including the short ones) contained an intron ofapproximately 100 bp. Four unique different clones out of the 20sequenced were identified. Two of them [SEQ6C(H64NORU1/W151) andSEQ7C(H64NORU2/UP3)] had an additional 20 aa (compared to H64MUT) butstill contained a stop codon located immediately at the beginning of thepeptide they formed. Other two fragments [SEQ8C(H64NORU3/UP16) andSEQ9C(H64NORU4/UP1)] did not contain any stop codon and were mostsimilar to the sequence of H64VES. These fragments added 26 aa to theH64MUT sequence and they both contain the two arginine residues as inH64VES which are most often found in the plastidic targeting signal ofmonoterpene synthases (see FIG. 3A).

Cloning H64MUT/H64NORS for Expression in E. coli

The E. coli expression vector pRSETB (Invitrogen) was used forheterologous expression of strawberry terpene synthases (see FIG. 5).The pRSETB vector contains the T7 promoter which can be induced byisopropyl-β-D-thiogalactopyranoside (IPTG) and therefore by insertingthe desired gene downstream of this promoter, the gene can be expressedin E. coli. In addition, DNA inserts were positioned downstream and inframe with a sequence that encodes an N-terminal fusion peptide. Thissequence includes (in 5′ to 3′ order from the N-terminal to C-terminal),an ATG translation initiation codon, a series of six histidine residuesthat function as a metal binding domain in the translated protein, theAnti-Xpress epitope, and the enterokinase cleavage recognition sequence.

The original pRSETB was primarily used for the insertion of the geneencoding the Green Fluorescent Protein (GFP). The GFP gene was fused tothe pRSETB vector using the BamHI and HindIII restriction sites locatedat the multiple cloning site (MCS) as can be seen in FIG. 5. Thisconstruct for the expression of GFP served as control for theexperiments together with the empty pRSETB vector.

Cloning the GFP gene to the PRSETB vector inserted an additional SalIrestriction site at the 3′ of the GFP gene and together with the BamHIsite located at the 5′ of the GFP gene served as sites for cloningH64MUT. The BamHI and SalI sites were introduced to the 5′ and 3′respectively of the H64MUT coding sequence by the use of PCR. The 552amino acid open reading frame of the H64MUT clone was amplified with thepfu DNA polymerase (Stratagene) and oligonucleotides (containing theBamHI and SalI sites) AAP339 (5′-CGGATCCGGCATC-GTCTTCTCGGGC-3′ SEQ IDNO: 8))and AAP334 (5′-CGTCGACCAACTCCACTTCCGGTAGTC-3′ SEQ ID NO: 9))according to the manufacturers instructions. The PCR product was clonedinto PCR-script vector (Stratagene), cut out with BamHI and SalI andfurther inserted (as a translation fusion) into the correspondingrestriction sites in the pRSETB vector. H64NORS was cloned in a similarway.

Bacterial Expression and Partial Purification Using the His Tag Columns.

The pRSETB vector harboring the H64MUT or H64NORS was used to transformE. coli strain BL21 Gold DE3 pLysE (Stratagene) as described by themanufacturer. For bacterial expression typically 1 ml of overnightliquid culture grown at 37° C. in Luria Broth (LB) medium (10 g/ltryptone, 5 g/l yeast extract, 5 g/l NaCl) supplemented with 100 mg/lampicillin was diluted 50 times in the same medium and grown until theOD₆₀₀ reached 0.4 (at 37° C.). At this stage IPTG was added to a finalconcentration of 1 mM in order to induce expression. After overnightgrowth at 16° C. the cells were harvested by centrifugation at 4000×gfor 15 min. Pellet and a sample from the supernatant were kept for SDSgel analysis. The cells were further processed as described by theNi-NTA Spin Columns manufacturers (QIAGEN) for protein purificationunder native conditions. First elute from the column (200 μl) wasfurther used for enzymatic activity assays.

Example 5 Cloning and Characterization of SOS Genes from Wild andCultivated Strawberry

Cloning of the SOS cDNA from Cultivated Strawberry (SOSA) and itsHomolog from the Wild Strawberry (SOSV)

For cloning the SOSA(MA) cDNA from the cultivated strawberry CV Elsanta,we designed an oligonucleotide on a published sequence of asesquiterpene cyclase from the wild strawberry (Nam et al. Plant Mol.Biol. 39: 629-636, 1999). The oligonucleotide (AAP 272,5′-GATGATATGTATGATGCATTCGG-3′ SEQ ID NO: 10)) was used to perform a 3′RACE reaction using the RACE kit (Clontech) and a 991 bp fragment wascloned. For cloning the full length cDNA we performed a 5′ RACE reactionusing an oligonucleotide designed on the 3′ UTR of the cDNA (AAP283,5′-GAAAGGATAGGCTCATCAGTACGTG-3′ SEQ ID NO: 11)). The entire SOSA(MA)cDNA cloned is 2605 bp long. We however could not identify an ORFencoding a protein longer then 255 aa, which is less then a half of atypical terpene synthase. Therefor a second attempt to clone a cDNA witha longer ORF was performed. Using oligonucleotides based on the SOSA(MA)sequence, one located on the beginning of the ORF (AAP325,5′-CGGATCCGCCTGTCCATGCTACTCC-3′ (SEQ ID NO: 12)) and the other on theUTR (AAP341, 5′-CGTCGACTGAGTTCAGAGTGGCACTGG-3′SEQ ID NO: 13)), a secondfull-length SOSA cDNA was isolated by the means of PCR on the cultivatedstrawberry cDNA [termed SOSA(WS)]. Sequencing SOSA(WS) revealed that asfor SOSA(MA) it contains a truncated ORF. We decided to clone the fulllength SOS homolog from the wild strawberry in order to identify thecause for such a truncation in the cultivated genes ORF. Cloning of thewild SOS homolog was performed by 3′ RACE reaction using anoligonucleotide designed on the SOSA(MA) ORF (AAP325, see above). Thefull length SOS homolog from the wild strawberry (SOSV) is 1973 bp longand contains a ORF encoding a 556 aa long protein. Aligning SOSA(MA),SOSA(WS) and SOSV nucleic acid sequences revealed minor changes in theORF (see FIG. 6). We could however identify the basis of the truncationin the cultivated SOS genes which was an insertion of two cytosinenucleotides causing a frame shift followed by a stop codon (see FIG. 6).Removing the CC insertion from the SOSA(WS) and SOSA(MA) genes resultsin the formation of ORFs encoding 554 and 555 aa respectively (FIG. 7).

PCR on Cultivated and Wild Strawberry Genomic DNA

In order to confirm the presence of the CC frame shift, causing atruncation in the cultivated strawberry SOS genes we analyzed theexistence of the insertion at the DNA level. PCR on both wild andcultivated strawberry genomic DNA was performed using twooligonucleotides located from both sides of the place of insertion(AAP345, 5′-AGAGGTTAGGTGCTCGGCGTTAC-3′ SEQ ID NO 14)) and the reverseoligonucleotide, AAP346, 5′ GAACAACTCCACGATCCTATCTC-3′ SEQ ID NO 15)).The expected amplified DNA fragment was 200 bp. PCR products at the sizeof 300 bp were obtained from both reactions using the wild andcultivated DNA. We sequenced 20 and 15 fragments from the cultivated andwild strawberry reactions respectively. All fragments contained anintron of approximately 100 bp. Sequence alignment of all fragmentsrevealed 7 different sequences from the cultivated and 5 from the wild.FIG. 8 shows an alignment of all fragments of the SOS genes both fromthe wild and cultivated strawberry obtained either from RNA (thedifferent cDNAs) or from DNA. Among the cultivated fragments we couldidentify 2 fragments which showed the CC insertion while the other 5 didnot contain it. On the other hand no fragment in the wild strawberrycould be detected that contained the frame shift mutation.

Analysis of SOS Expression in Ripe Cultivated and Wild Strawberry Fruit

Using the SOSV cDNA as a probe we analyzed SOS gene expression in twodifferent wild and cultivated cultivars (FIG. 9). The SOSV cDNA could beused for hybridization with blots containing RNA from both wild andcultivated cultivars since the SOSA genes and SOSV share nearly 99%identity at the nucleic acid level (in the ORF region). Hardly anyexpression could be detected in the cultivated cultivars while strongexpression could be detected in the wild cultivars. The SOSA probe wasalso used for hybridizing blots with RNA extracted from differentcultivated (Elsanta) fruit developmental stages, but just weak signalcould be detected after long exposure. Nam et al., (1999) were also notable to detect expression of the partial cDNA homolog of SOS with RNAderived from different fruit developmental stages of the cultivatedstrawberry. Expression in different wild strawberry plant tissues wasrestricted to the fruit, specifically to the red ripe stage.

Cloning and Expression of SOSV and SOSA in E. coli

Both the SOSA and SOSV coding regions were used for the formation of arecombinant protein in E. Coli cells. The entire ORF of SOSA cDNAalthough truncated was expressed in order to serve as a negative controlfor the enzymatic assays. Similar to the cloning of H64MUT the BamHI andSalI restriction sites at the 5′ and 3′of the GFP gene respectivelyserved as sites for the cloning of SOSA and SOSV ORFs into the pRSETBexpression vector. The BamHI and SalI sites were introduced to the 5′and 3′ respectively of the wild and cultivated SOS genes coding sequenceby the use of PCR. The restriction sites were added to theoligonucleotides used for PCR reaction (AAP325,5′-CGGATCCGCCTGTCCATGCTACTCC-3′ (SEQ ID NO: 12) and the reverse primerAAP341, 5′-CGTCGACTGAGTTCAGAGTGGCACTGG-3′). The PCR product was clonedinto PCR-script vector (Stratagene), cut out with BamHI and SalI andfurther inserted (as a translation fusion) into the correspondingrestriction sites in the pRSETB vector. Expression of SOSA and SOSV inE. Coli was performed parallel to the expression of H64MUT and underidentical experimental conditions.

Example 6 Analysis of SOSA, SOSV, H64MUT and H64NORS Recombinant Enzymes

For determination of terpene synthase identity, the His-tag purifiedenzymes (prepared as described above under Example 4.6) were diluted10-fold with buffer A containing 15 mM MOPSO (pH 7.0), 10% glycerol, 10mM MgCl₂, 1 mM sodium ascorbate and 2 mM DTT. To 1 mL of this enzymepreparation, 40 μM of either [³H]-geranyl diphosphate (GPP) or[³H]-farnesyl diphosphate (FPP) were added. Assays with GPP as substratewere also supplemented with 1 mM MnCl₂. After the addition of a 1-mLredistilled pentane overlay, the tubes were carefully mixed andincubated for 1 h at 30° C. After the assay, the tubes were vortexed,the pentane layer was removed and passed over a short column of aluminumoxide overlaid with anhydrous Na₂SO₄. The assay was re-extracted with 1mL of diethyl ether, which was also passed over the aluminum oxidecolumn, and the column washed with 1.5 mL of diethyl ether. 100 μL ofthe organic exrtract was removed for liquid-scintillation counting in4.5 mL of scintillation cocktail (Ultima Gold, Packard Bioscience, TheNetherlands). Radio-labelled products were present in the organicextracts of:

H64MUT H64NORS SOSV SOSA [³H]-GPP + + + − [³H]-FPP + + − −

Subsequently, the extracts were carefully concentrated under a stream ofN₂ before analysis using radio-GLC and GC-MS. Radio-GLC was performed ona Carlo-Erba 4160 Series gas chromatograph equipped with a RAGA-90radioactivity detector (Raytest, Straubenhardt, Germany). Samplecomponents eluting from the column were quantitatively reduced beforeradioactivity measurement by passage through a conversion reactor filledwith platinum chips at 800° C. Samples of 1 μL were injected in the coldon-column mode. The column was a fused silica capillary (30 m×0.32 mmi.d.) coated with a film of 0.25 μm of polyethylene glycol (EconoCapEC-WAX, Alltech Associates) and operated with a He-flow of 1.2 mL min⁻¹.The oven temperature was programmed to 70° C. for 1 min, followed by aramp of 5° min⁻¹ to 210° C. and a final time of 10 min. About 20% of thecolumn effluent was split with an adjustable splitter to an FID(temperature 270° C.). The remainder was directed to the conversionreactor and radio detector. H₂ was added prior to the reactor at 3 mLmin⁻¹, and CH₄ as a quench gas prior to the radioactivity detector (5 mLcounting tube) to give a total flow of 36 mL min⁻¹. Radio-GLC analysisgave the following results:

-   -   the SOSV and H64MUT and H64NORS recombinant proteins catalysed        the formation of radio-labelled products from [³H]-GPP (FIG.        10). For the SOSV protein a number of radio-labelled product        peaks were visible in the retention time area of olefinic        monoterpenes (FIG. 10B). The major radio-labelled product did        not co-elute with any of the added unlabelled reference        compounds, but one of the minor radio-labelled peaks seemed to        co-elute with the reference β-myrcene. For the H64MUT        recombinant enzyme the single radio-labelled product co-eluted        with linalool (FIG. 10C).    -   with [³H]-FPP as substrate scintillation counting showed that        neither the SOSA nor the SOSV recombinant protein catalysed any        radio-labelled product formation. The H64MUT protein catalysed        the formation of a radio-labelled product which radio-GC        analysis showed to be one single product, co-eluting with        trans-nerolidol (FIG. 11).

The samples were also analysed by GC-MS using a HP 5890 series II gaschromatograph equipped with an HP5-MS column (30 m×0.25 mm i.d., 0.25 μmdf) and HP 5972A Mass Selective Detector (Hewlett-Packard). The oven wasprogrammed at an initial temperature of 45° C. for 1 min, with a ramp of10° C. min⁻¹ to 280° C. and final time of 5 min. The injection port(splitless mode), interface and MS source temperatures were 250, 290 and180° C., respectively, and the He inlet pressure was controlled byelectronic pressure control to achieve a constant column flow of 1.0 mLmin⁻¹. Ionization potential was set at 70 eV, and scanning was performedfrom 48-250 amu. The m/z 93 chromatogram of SOSV recombinant proteincatalysed products from [³H]-GPP again shows several peaks (FIG. 12) aswas also seen in the radio-GC chromatogram (FIG. 10B). The compoundswere identified as α-pinene (major compound), β-pinene, sabinene,β-myrcene, α-phellandrene, β-phellandrene, dihydromyrcenol (tentative),α-terpinolene (tentative) and α-terpineol (tentative). This shows thatSOSV is not a sesquiterpene synthase as is claimed for a fragmentnucleic acid isolated by Nam et al (Plant Mol Biol, 39: 1999-2002, 1999)and Marty (EMBL Database, Accession number AJ001452), but a monoterpenesynthase, viz. an α-pinene synthase. Nam et al and Marty had isolatedjust a fragment of the cDNA and for example missed the 5′-side. Hence,the authors were also not able to functionally express the protein andidentified it wrongly as a sesquiterpene synthase. The GC-MSchromatograms of the incubations of the H64MUT protein with [³H]-GPP or[³H]-FPP show the presence of one terpene product for each substrate andcomparison of the retention times and mass spectra with authenticstandards confirmed that from [³H]-GPP linalool was produced (FIG. 13)and from [³H]-FPP trans-nerolidol (FIG. 14). Analysis usingenantioselective columns showed that both linalool and nerolidol were ofthe S configuration, so (3S)-(E)-nerolidol and S-linalool.Characterisation. The H64NORS encoded and his-tag purified protein wasshown to have an optimum pH of around 7 for both GPP and FPP. For bothsubstrates there was no preference for Mn²⁺ (at 1 mM) or Mg²⁺ (at 10 mM)and therefore a combination of the two was routinely used. The affinityof the enzyme for the two substrates strongly differed. The Km for FPPwas 3.2 μM which is in the expected range for sesquiterpene synthases.However, for GPP the Km was >50 μM which is highly unusual. However, theapparent Vmax for GPP was much higher than for FPP.

Example 7 Analysis of Targeting

We used transient expression assays using the Green Fluorescent Protein(GFP) to identify the sub-cellular localization of the proteins encodedby the different nucleic acid fragments described in this invention(FIG. 15). We first constructed 13 different constructs which fusedin-frame the 5′-end parts of the different genes (H64NORL, H64NORS,H64TAR4, H64VES, SOSV) to the GFP gene (FIG. 16). Different regions ofthe 5′-ends were used part of them included a portion from the proteinitself (up to the MID motif). Expression in plants was driven by the 35Scauliflower mosaic virus promoter. Plasmid DNA from constructs was usedto transform tobacco protoplasts. After transformation the protoplastswere incubated for 24 hr at 28° C. in the dark and thereafter used forthe analysis of GFP transient expression and subcellular localizationusing confocal laser scanning microscopy. The results demonstrated thatthe 5′-ends of both H64TAR4 and H64VES encode a targeting signal (FIG.15). The protein encoded by H64TAR4 is targeted to the plastids (e.g.chloroplasts) and mitochondria while the H64VES protein is targeted tothe plastids (e.g. chloroplasts). H64NORL and H64NORS, which are mostactive in the ripe cultivated strawberry, are targeted to the cytosol.SOSV is also targeted to the cytosol, in-contrast to all monoterpenesynthases described to date which are plastid localized. Thus, accordingto this experiment for monoterpene synthases the cytosol and not onlythe plastids are a possible location and in the cytosol there are highlevels of GPP to synthesize the monoterpenes. For sesquiterpenesynthases normally reported to be localized in the cytosol othersub-cellular localization may be possible such as in the mitochondriaand chloroplasts and they may use FPP in these compartments and producehigh levels of the sesquiterpene. We also demonstrated by the samemethod that the different targeting signals of the terpene synthasescould be easily swapped by the use of site-directed mutagenesis. Forexample the plastidic targeting signal encoded by the H64VES N-terminalpart could be modified to dual targeting to mitochondria andchloroplasts by a change in 2 amino acid residues (Tryptophan-W6 changedto Arginine-R6 and deletion of Isoleucine-116).

Example 8 Effects of Nerolidol on Agrobacterium tumefaciens

FPP, the precursor for sesquiterpene biosynthesis is a most commonmetabolite and exists in every living organism. Thus, the expression ofa protein encoding a nerolidol synthase will result in the conversion ofendogenous FPP to nerolidol in most living organisms. We constructed abinary vector (plasmid used for the transformation of plants cells,which lacks the virulent genes present on the Ti plasmid of the virulentstrain of Agrobacterium tumefaciens) containing the H64NORS gene flankedby a 35S CaMV promoter (5′-end) and a Nopaline Synthase (NOS) terminator(3′-end) and used it to transform 2 different strains of Agrobacterium.In both cases no colonies were obtained after plating the transformationreaction on Luria Broth (LB) medium containing 50 mg/l kanamycin andRifampicin. Thus, the H64 NORS gene was expressed in Agrobacterium andthe protein encoded by it converted the bacterial endogenous FPP tonerolidol, which is highly toxic to the Agrobacterium cells, andtherefore no transformants were obtained. Thus, transgenic plantsexpressing a nerolidol synthase will have an anti-microbial effect andcould be used for the protection against Agrobacterium crown-galldisease. In order to be able to introduce a plasmid containing such aterpene synthase having toxic effects on the bacteria one can introduceone or more introns into the coding sequence of the gene. These intronscan not be spliced by the bacteria and hence no functional protein isformed by the micro-organism. In the plant, the normal eukaryoticsplicing process will lead to a functional protein. The introduction ofsuitable, organ-specific and/or inducible promoters in the appropriateconstruct will allow the directed expression of linalool and/ornerolidol at the appropriate site to control crown-gall disease inplants such as fruits, rose, etc. Also, slow release formulations orother compositions containing linalool and/or nerolidol may be useful tocontrol crown-gall disease.

Example 9 Effects of Linalool and Nerolidol on Spore Germination, LesionGrowth and Sporulation of Phytophthora infestans, Fusarium spp. andBotrytis spp.

Comparison of Effects of Farnesol and Linalool on Mycelium Growth ofPhytophthora infestans on Growth Medium

Farnesol and linalool were tested in two concentrations (2% and 0.2%(v/v)) through the addition to Plich medium in 6 well plates (3 ml perwell). One 6 well plate per compound was used with two differentconcentrations in triplicate. All wells were inoculated with a plug ofPhytophthora infestans mycelium (isolate VK98014, 1 month old) andincubated at 20° C. On day 3, 5 and 7 the radial growth of the myceliumwas measured. An overview of the results is given in FIG. 17. Themycelium growth of Phytophthora infestans was inhibited completely 3, 5and 7 days after the experiment by both the high and the lowconcentrations of linalool. Farnesol resulted in a partial inhibition ofmycelium growth at both the high and the low concentration. Theexperiment demonstrates that linalool is more active than farnesol forthe inhibition of mycelium growth of Phytophthora infestans.

Comparison of the Effects of Linalool and Nerolidol on Mycelium Growthof Phytophthora infestans Alone and in Combination on Growth Medium

Linalool and nerolidol were tested in three concentrations (0.2%, 0.02%and 0.002% (v/v)) through the addition to Plich medium in 6 well plates(3 ml per well). One 6 well plate per compound was used with twodifferent concentrations in triplicate. To study whether the compoundsacted directly or through the vapour phase in one plate mycelia weregrown on control medium with the compounds (0.2%) added to the medium inthe adjacent wells. Free exchange of the compounds through the vapourphase was possible this way. All wells were inoculated with a plug ofPhytophthora infestans mycelium (isolate VK98014, 1 week old) andincubated at 20° C. On day 3 and 5 the radial growth of the mycelium wasmeasured. The results are shown in FIGS. 18-21.

FIG. 18 shows that linalool is active even at the lowest concentrationof 0.002% (=20 ppm). Remarkably, the effects of linalool are equallyeffective through the. vapour phase as through the medium. Apparentlythis monoterpene is so volatile that the active concentrations in thevapour and medium phase are similar. This high activity in the vapourphase makes linalool an attractive compound for the protection of storedproducts against micro-organisms e.g. the protection of potato toPhytophthora, Phoma, and Fusarium.

FIG. 19 shows that nerolidol is slightly more effective than linalool(compare FIG. 18) in inhibiting Phytophthora infestans mycelium growthand that it is a strong inhibitor of mycelium growth even at the lowestconcentration of 20 ppm. In contrast to linalool the effects through thevapour phase are negligible. This can be explained by the fact that thesesquiterpene nerolidol is much less volatile than the monoterpenelinalool.

FIGS. 20 and 21 show that the action of linalool and nerolidol isstronger in combination than when taken alone. This suggests that thesimultaneous production of these compounds in plants could result inmore effective fungal control compared to a situation when only one ofthe two compounds is present.

The effect of Nerolidol Infiltrated in Potato Leaves on the Germinationof Phytophthora infestans Spores, Lesion Formation and Sporulation.

Leaves of potato cultivar Bintje were vacuum-infiltrated with a 0.05%(v/v) solution of nerolidol. This was done by placing 6 leaflets at atime in a 50 ml bluecap with the nerolidol solution or a water control.The tubes were placed for 15-30 min under vacuum which was then suddenlyreleased. Good infiltration was visible by the dark green color of theleaves. The leaves had gained about 25% weight this way so that theactual concentration in the leaves was in the range of ca. 0.0125%. Theleaves were placed on water agar (1.5%) and inoculated with 250-500spores of the 4 different Phytophthora infestans isolates (race-0,IPO-c, 428-2, VK98014). The leaves were incubated one night in the darkat 15° C. and then moved to normal lighting conditions (15° C., 16hlight, 8h dark). After 7 days the leaves were scored for the formationof lesions and sporulation. The results demonstrate that also wheninfiltrated in potato leaves nerolidol strongly inhibits myceliumgrowth, lesion formation and sporulation at a low concentration. Theeffects appear to be not race-specific but equally affecting the fourdifferent isolates showing that also nerolidol provides broad resistanceagainst this fungus.

Effects of Linalool and Nerolidol on Fusarium and Botrytis mycelialGrowth

Fusarium. Nerolidol and linalool were tested in a range ofconcentrations (10-5000 ppm) alone and in combination through theaddition to Plich medium in 6 well plates (3 ml per well). In the caseof comparing the application of nerolidol and linalool alone to thecombination of the two compounds, 100 ppm of the single compound was forexample compared to 50+50 ppm of the two compounds together. One 6 wellplate per compound was used with two different concentrations intriplicate. All wells were inoculated with a mycelium plug of Fusariumgraminearum, Fusarium culmorum or F. verticillioides strain MRC826 andincubated at 20° C. Each day the radial growth of the mycelium wasmeasured. The results of day 7 are given in FIG. 22. The mycelium growthof all Fusarium spp. was inhibited at concentrations above 10 ppm. Atlow concentrations nerolidol was slightly more effective than linaloolin the case F. graminearum and MRC826. At very high concentrationslinalool was more effective. The combined use of nerolidol and linaloolis at least as effective as either individual compound and appears toprovide a more robust inhibition against all Fusarium species.

Botrytis. Nerolidol and linalool were tested in a range ofconcentrations (10-5000 ppm) alone and in combination through theaddition to Plich medium in 6 well plates (3 ml per well). In the caseof comparing the application of nerolidol and linalool alone to thecombination of the two compounds, 100 ppm of the single compound was forexample compared to 50+50 ppm of the two compounds together. One 6 wellplate per compound was used with two different concentrations intriplicate. All wells were inoculated with a mycelium plug of Botrytiscinerea isolated from grape and strawberry and incubated at 20° C. Eachday the radial growth of the mycelium was measured. The results of day 7are given in FIG. 23. The mycelium growth of all Botrytis isolates wasinhibited at concentrations above 10 ppm. At low concentrationsnerolidol was more effective than linalool. At very high concentrationslinalool or the combination of linalool and nerolidol was mosteffective. The combined use of nerolidol and linalool is at least aseffective as either individual compound and appears to provide the mostrobust inhibition against all Botrytis isolates.

Effects of Linalool and Nerolidol on Fusarium Spore Germination.

A spectrophotometric assay was used to monitor the onset of germinationof Fusarium verticillioides spores (isolates: ITEM2282, MRC3235, MRC826,MRC826-2) in solution. The spores were diluted to a concentration of 10⁵spores/ml in Czapek Dox medium and mixed with 8-4000 ppm of linalool ornerolidol. Linalool did not affect the germination of the spores at all(FIG. 24B). Nerolidol, however, showed strong inhibition of germinationat concentrations above 250 ppm (FIG. 24A). This suggests that nerolidolprovides an additional mode of control of Fusarium verticillioides atthe level of spore germination in comparison to linalool and that forthe most effective control at all stages of fungal development ofFusarium spp a combined use of nerolidol and linalool is mostappropriate.

Example 10 Transformation and Characterization of Arabidopsis, Potato,Tomato and Petunia

Preparation of Constructs for the Transformation of Plants.

Constructs with the H64 cDNAs are prepared for the transformation ofplants in order to yield plants that will produce linalool and/ornerolidol in variuous compartments:

The cDNAs are placed under the control of either the 35S promoter or theRubisco promoter, both separately and in combination in order to obtainplants producing linalool or nerolidol alone or in combination. It isalso contemplated that for some purposes glycosylation ordeglycosylation of the terpene-alcohol is required for the mode ofaction against fungi or insects. For this reason also constructs aremade containing glycosyl-transferases or glycosidases in conjunctionwith the linalool and/or nerolidol synthase cDNAs.

Construction of Binary Vectors.

The appropriate sequences were ligated into a pFlap 10 vector. Theligation product was transformed to E. coli DH5α competent cells, andtransformed colonies were grown O/N at 37° C. and 250 rpm. Theexpression cassette was removed from the resulting vector by using PacIand AscI restriction enzymes (NEB, England) and ligated into the binaryvector pBINPLUS, containing a kanamycin resistance selection marker(nptII), after digestion with PacI and AscI. Colonies were checked aftertransformation by back-transformation to E. coli DH5α competent cells.

Transformation of Arabidopsis

We used the floral-dip transformation method to transform Arabidopsisplants ecotype Columbia according to Marsh-Martinez et al. (2002). Aftercollecting the seeds they were let to dry for several days and then sownon MS medium containing 50 mg/l kanamycin and 400 mg/l cefotaxime. Greenshoots, 1 cm in size were transferred to the green house and grown tomaturity.

Transformation of Potato

On day 1 an Agrobacterium tumefaciens culture of AGLO containing aBINPLUS derived binary vector was started in 50 ml LB-medium containing50 mg/l kanamycin and shaken for 2 days at 28° C. On day 2 internodesfrom an in vitro culture of the potato cultivar Desiree were cut into0.5-1 cm pieces and placed on R3B medium (30 g/l sucrose, 4.7 g/lMurashige and Skoog salts, pH 5.8 (KOH), 8 g/l purified agar, 2 mg/l NAAand 1 mg/l BAP) which was covered with 2 sterile filterpapers that hadpreviously been soaked in 2 ml PACM medium (30 g/l sucrose, 4.7 g/lMurashige and Skoog salts, 2 g/l casein hydrolysate, pH 6.5 (KOH), 1mg/ml 2,4-D and 0.5 mg/l kinetine). The dishes were taped with parafilmand incubated overnight at 24° C. under a regime of 16 h light. At day 3the A. tumefaciens culture was poured in a sterile petridish containingthe explants. After 5-10 min explants are removed from the culture,placed on a sterile filter paper to remove excess Agrobacteria andplaced back on the R3B medium containing dishes after first removing thetop filter paper (leaving one behind). Dishes with explants were furtherincubated at 24° C. and 16 h light until day 5, when the explants weretransferred to dishes containing ZCVK medium (20 g/l sucrose, 4.7 g/lMurashige and Skoog salts, pH 5.8 (KOH), 8 g/l purified agar, 1 mg/lzeatine, 200 mg/l vancomycin, 100 mg/l kanamycin, 200 mg/l claforan). Onday 19 and subsequently every 3-4 weeks explants were transferred to newZCVK medium. When shoots appeared shoots were transferred to Murashigeand Skoog medium containing 20% sucrose (MS20). After rooting plantswere transferred to the green house

Petunia Transformation.

Leaf cuttings of Petunia W115 were transformed with Agrobacteriumtumefaciens strain LBA4404 using a standard plant transformationprotocol (Lücker et al., The Plant Journal 27: 315-324, 2001). As acontrol leaf cuttings were also transformed with LBA4404 containing thepBINPLUS vector. Furthermore some non-transformed leaf cuttings werecarried through the regeneration process. Rooting plants, arising fromthe Agrobacterium transformation were tested with PCR for the presenceof the respective gene construct. Positive plants were transferred tothe greenhouse. All ransgenic plants were phenotypically normal andshowed a normal development compared with non-transformed controlplants, which had gone through the same regeneration process.

Tomato Transformation.

The tomato cultivar ‘Micro-Tom’ (Lycopersicon flavour) was used (Scottand Harbaugh, 1989). The plants were grown from seeds provided by a seedcompany (Beekenkamp seed, Holland). Micro-Tom seeds were firststerilised. A rinse in 70% ethanol followed by a two hour bleaching in1.5% HClO₄. After bleaching, the seeds were quickly rinsed in watertwice and then washed in water for ten and sixty minutes. Aftersterilisation, seeds were sowed in pots, containing 80 ml vermiculiteand 70 ml of germination medium containing 4.4 g/l MS salts withvitamins and 0.5% sucrose (pH 5.8).

After 7 days of growth in a culture room (25° C.), covered with 2 foldsof filter paper, the cotyledons were cut under water near the petioleand the tip with a rolling action of the scalpel, to minimize damage.The explants were placed on their backs on filter paper on feederlayersto incubate overnight in the culture room (25° C.), covered with 4 foldsof filter paper, under low light conditions. After incubation, theexplants were immersed in the Agrobacterium suspension for 20 minutes.After immersion, the explants were placed back on feederlayers forco-cultivation, following a rinse in a solution containing 400 mg/lcarbenicillin and 100 mg/l tricarcillin. The explants were placed incallus inducing medium (4.4 g/l MS salts with Nitsch vitamins, 3%sucrose, 0.8% purified agar (Oxoid), pH 6.0, 2 mg/l zeatin, 400 mg/lcarbenicillin, 100 mg/l tricarcillin, 100 mg/l kanamycin). The plateswere covered with 2 folds of filter paper and set to grow in a cultureroom (25° C.) under low light conditions for 3 weeks. Formed callus wastransferred to shoot inducing medium (as callus inducing medium, butwith 1 mg/l zeatin, 200 mg/l carbenicillin, 50 mg/l tricarcillin).

These plates were set to grow under the same conditions as thecallus-inducing plates. Shoots formed were transferred to rooting mediumin pots (4.4 g/l MS salts with Nitsch vitamins, 3% sucrose, 0.5% agargel(Sigma), pH 6.0, 0.25 mg/l IBA, 50 mg/l kanamycin, 400 mg/lcarbenicillin. The growing conditions remained the same. Fully-grownplants were subsequently transferred to the greenhouse.

Analysis of the Transgenic Plants with Capillary Gas Chromatography—MassSpectrometry (GC-MS).

The tissues to be analyzed were collected in the greenhouse and frozenin liquid nitrogen. In general, 200 mg frozen material was homogenizedand transferred to a mortar containing 1.5 mL 5M CaCl₂ and a smallamount of purified sea sand. These tissues were mixed with 0.75 mL 5MCaCl₂. The material was ground rapidly and thoroughly with a pestle,inhibiting enzymatic reactions. 0.75 mL of the material was introducedinto a 1.8 mL GC vial containing a small magnetic stirrer. The vial wasthen closed with an aluminum cap with a PTFE/Butylrubber septum.Subsequently the vial was placed in a 50° C. waterbath and preheated for20 minutes while stirring. The headspace sampled during 30 minutes witha 100μ PDMS SPME fiber (Supelco, Belfonte Pa. USA).

GC-MS analysis was performed using a Fisons 8060 gas chromatographdirectly coupled to a MD 800 mass spectrophotometer (Interscience,Breda, the Netherlands). A HP-5 column (50 m×0.32 mm, film thickness1.05 μm) was used with He (37 kPa) as carrier gas. GC oven temperaturewas programmed as follows: 2 min 80° C., ramp to 250° C. at 8° min⁻¹ and5 min 250° C. Mass spectra in the electron impact mode were generated at70 eV. The compounds were identified by comparison of GC retentionindices and mass spectra with those of authentic reference compounds.Injection was performed by thermal desorption of the SPME fiber in theinjector at 250° C. during 1 min using the splitless injection mode withthe split valve being opened after 60 sec. Alternatively, volatiles weretrapped on cartridges containing Tenax, eluted using pentane/ether andanalysed using GC-MS essentially as described by Bouwmeester et al(1998). Transgenic Arabidopsis plants expressing H64TAR, for example,produced large amounts of linalool and smaller amounts of nerolidol(FIG. 25). Transgenic potato lines also produced substantial amounts oflinalool, but also the hydroxy-derivative 8-hydroxylinalool (FIG. 26).Interestingly, the native linalool of potato, which can also bedetected, had a different stereochemistry as the transgenic linalool(FIG. 26), which allowed a clear distinction between native andtransgenic product. Because it was suspected that in some of the plantspecies these compounds were present in a bound form, leaf material ofPetunia (transgenic and control samples) was harvested and frozen inliquid nitrogen, and ground to a fine powder in a cooled mortar andpestle. In total 60 mg of the powdered leaf material was transferred to0.5 ml of citrate buffer at pH 4.5, to which 140 i.u. β-glucosidase wereadded. The vial was capped and incubated during 12 h at 25° C.Subsequently, the headspace of the vial was sampled during 30 minuteswith 100 micron PDMS solid phase microextraction device and analysedusing GC-MS as described above. No linalool or nerolidol was detectablein samples from the untransformed control plants, whereas in thetransgenic plants both linalool and nerolidol were detected. The sampleof transgenic leaf material without beta-glucosidase present during theincubation did not show any detectable linalool or nerolidol, indicatingthat all linalool and nerolidol is stored in the petunia leaves in theform of its glucoside, instead of continuous emission as was describedfor linalool in the flowers of Clarkia breweri.

Identification of Glycosides in Transgenic Plants

High-performance-liquid-chromatography electrospray-ionization tandemmass spectrometry (HPLC-ESI-MS-MS) analysis of methanol extracts wasperformed on a triple stage quadrupole TSQ 7000 LC-MS-MS system with anelectrospray ionization (ESI) interface (Finnigan MAT, Bremen, Germany).The temperature of the heated capillary was 240° C. The ESI capillaryvoltage was set to 3.5 kV, resulting in a 3.4 μA current. Nitrogenserved as both the sheath (70 psi) and auxiliary gas (10 L/min). Dataacquisition and evaluation were carried out on a Personal DECstation5000/33 (Digital Equipment, Unterfohring, Germany) and ICIS 8.1 software(Finnigan MAT). HPLC separation was carried out on an Eurospher 100 C-18column (100×2 mm, 5 μm, Knauer, Berlin, Germany) using a linear gradientwith a flow rate of 200 μL min⁻¹. Solvent A was 5 mM ammonium acetate inwater, and solvent B was 5 mM ammonium acetate in methanol. The gradientprogram was as follows: 0-30 min 5 to 100% B.Mass spectra were acquiredin the negative mode. Product ion spectra were available bycollision-induced dissociation (CID) (1.5 mTorr of Argon; −20 eV). Forpreparation of extracts plant leaves (3 to 7 g) were homogenized in 50ml of 80% methanol and centrifuged (2000 g for 5 min). The residue waswashed with 50 ml of 80% methanol and the supernatants were combined.Methanol was removed in vacuum and the remaining aqueous solution wasextracted with 2×20 ml diethyl ether. The extract was subjected to XAD-2(20 cm, 1 cm inner diameter) solid phase extraction. The column wassuccessively washed with 50 ml water and 50 ml diethyl ether. Glycosideswere eluted with 80 ml methanol. The extract was concentrated in vacuum.The residue was dissolved in 1 ml of 50% methanol in water and analyzedby HPLC-ESI-MS-MS.

R,S-Linalyl β-D-glucopyranoside was synthesized from R,S-linalool and2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl bromide according to amodified Koenigs-Knorr synthesis. For enzymatic hydrolysis an aliquot ofthe methanol extract was dissolved in 2 ml of 0.2 M phosphate buffer (pH5.5), and 200 μl of Rohapect D5L (Röhm, Darmstadt, Germany), apectinolytic enzyme preparation exhibiting glycosidase activity wasadded. After an incubation period of 24 h at 37° C., the liberatedaglycons were extracted two times by 1 ml of diethyl ether each. Thecombined organic layers were dried over Na₂SO₄ and concentrated.Multidimensional gas chromatography mass spectrometry (MDGC-MS) analyseswere performed with tandem Fison 8160 GC connected to a Fison 8130 GCand a Fisons MD 800 quadrupole mass spectrometer equipped with FisonsMassLab software (Version 1.3). The first GC was fitted with a splitinjector (1:10, at 230° C.) and a flame ionization detector (at 250°C.). The first GC employed a 25 m×0.25 mm i.d. fused silica capillarycolumn coated with a 0.25 μm film of DB-Wax 20 M (J & W Scientific) forthe pre-separation of the target molecule. Separation of enantiomers wasachieved with the second GC using a 25 m×0.25 mm i.d. fused silicacapillary column coated with a 0.15 μm film of 2,3-di-O-ethyl-6-O-tert.Butyl dimethylsilyl-β-cyclodextrin/PS086. The column in GC1 wasconnected by a multicolumn switching system (Fisons) to the column inGC2. The retention time of the compound of interest was determined by GCseparation while the column in GC1 was connected to the FID. Separationof the enantiomers was achieved in the second GC after transfer of thecompound of interest from the capillary column in GC1 to the column inGC2 via the switching device. The fused silica capillary column in GC1was maintained at 60° C. then programmed to 240° C. at 10° C. min⁻¹ withHe gas flow at 3 ml min⁻¹. The fused silica capillary column in GC2 wasmaintained at 60° C. (15 min) then programmed to 200° C. at 2° C. min⁻¹with He gas flow at 3 ml min⁻¹. The compound of interest was transferredfrom GC1 to GC2 from 9.8 min to 10.3 min. The MS operating parameterswere ionization voltage, 70 eV (electron impact ionization); ion sourceand interface temperature, 230° C. and 240° C., respectively.Linalyl-β-D-glucopyranoside was synthesised in order to verify theidentity of the glycoside present in the transgenic petunia tissuetransformed with S-linalool synthase. HPLC-MS/MS analysis on control andtransgenic Petunia tissue as shown in FIG. 28, revealed that the m/z 375ion trace (FIG. 27A) of the compound detected in the transgenic Petuniatissue had the same retention time as one of the two diastereomers of(R,S)-linalyl β-D-glucoside that are slightly resolved in ion trace A.Also the product ion spectrum of the synthesised reference compound fitsthe spectrum of the peak detected in the transgenic petunia tissuenicely (FIG. 28B). The control Petunia tissue ion trace m/z 375 showedonly a slight elevation above background level at the retention time ofthe linalyl β-D-glucoside indicating that there is also a basal level oflinalyl-β-D-glucoside present in the plant before transformation (FIG.28A). Following Chiral phase Multidimensional Gas Chromatography MassSpectrometry (MDGC-MS) analysis, after enzymatic hydrolysis of theglucoside fraction of leaf tissue, revealed that the transgenic Petunialeaf contains highly enriched (S)-linalyl-β-D-glucoside. The controlplant however contains slightly enriched (R)-linalyl-β-D-glucoside.Since no tissue-specific promoter for expression was used, the enzymecan be formed in all plant organs and will give a product in all cellswhere GPP is present. By the action of a highly active endogenousglucosyltransferase of Petunia that is able to efficiently bind theS-linalool produced by the transgenic plants as(S)-linalyl-β-D-glucoside, cellular damage is prevented. Such a highlyactive glycosyltransferase was also reported in transgenic Kiwi fruitexpressing stilbene synthase, that accumulated picied(resveratrol-glucoside) in stead of resveratrol. Large-scalevolatilisation of linalool from the transgenic plants could be excluded,since only traces of linalool were detectable when the headspace of thetransformed plants was analysed. Volatilisation only occurred from theflowers and not from leaves. This in contrast to Arabidopsis where largeamounts of linalool were emitted from the leaves (FIG. 25). Therefore weconclude that most of the linalool in Petunia is directly bound as aβ-D-glucoside.

Further analysis of potato-leaf extracts also showed the presence ofglucosides, not only of linalool itself but also of 8-hydroxylinalool.In addition, more derivatives of linalool were found such aslinalool-triol, including the corresponding glucoside (Table 1).

In conclusion, transgenic plants expressing the inserted transgenes. areshown to produce the expected terpenoid compounds. Their amounts,release, oxidation to polyols, and derivatization to glycosides varyfrom species to species and can be influenced by the co-expression ofother sequences (see Example 11). When these compounds are not stored inany bound intermediates such as glycosides, the plants have alteredolfactory characteristics.

TABLE 1 control TM9 TM13 TM29 sample weight (g) 3.5 3.5 4.1 3.0 linalool0.3 6.6 4.0 10.5 (μg/g fresh weight) by GC-MS 8-hydroxylinalool 0.1 3.72.1 4.7 (μg/g fresh weight) by GC-MS linalooltriol <0.1 <0.1 <0.1 0.3(μg/g fresh weight) by GC-MS glycosidically bound 0.4 3.3 0.6 1.0linalool (μg/g fresh weight) by GC-MS glycosidically bound 1.6 18.7 8.927.2 8-hydroxylinalool (μg/g fresh weight) by GC-MS glycosidically bound<0.1 3.3 1.4 5.8 linalooltriol (μg/g fresh weight) by GC-MSlinalylglucoside 17 83 55 90 (μg/g fresh weight) by LC-MS 8- 12 101 51126 hydroxylinalylglucoside tentatively (μg/g fresh weight) by LC-MSlinalyltriolglucoside <1 39 24 38 tentatively (μg/g fresh weight) byLC-MS linalool 74:26 5:95 5:95 1:99 enantiomeric ratio (R:S) by MDGC-MSglycosidically bound 96:4  1:99 2:98 1:99 linalool enantiomeric ratio byMDGC-MS

Example 11 Effects of Changes in Targeting of Sesquiterpene Synthases toAchieve High Level Expression of Sesquiterpene Compounds inMitochondria.

It is commonly accepted that sesquiterpene biosynthesis in plants occursin the cytosol and not in any other cell compartment (Bick and Lange(2003) ABB 415: 146-154). It is also the current knowledge and this hasbeen described in several publications that the only isoprenoidsproduced by plants in the mitochondria are the prenyl chains ofubiquinones. The state of the art did not contain any teaching whichwould reliably predict that the expression of a cytosolic sesquiterpenesynthase in mitochondria would result in high expression ofsesquiterpene compounds. Indeed, the literature which did predict thatconstitutive sesquiterpene production could be achieved by expression ofcytosolic sesquiterpene synthases in the native location of the cytosolproved to be unreliable with absent or very low levels (thisapplication, Wallaart et al., Planta 212: 460-465, 2001; Hohn andOhlrogge, Plant Physiol 97: 460-462, 1991). Because of the presence of amitochondrial targeted FPP synthase in the Arabidopsis genome, weexpected that FPP, the substrate for the nerolidol synthase, would bepresent in the mitochondria of Arabidopsis, but it did not provide anyclue as to whether the substrate pool would be available tosesquiterpene synthases which are normally expressed in the cytosol, norto which quantity the sesquiterpenes would be produced.

In this example we present a method which depends on overexpressing acytosolic sesquiterpene synthase in mitochondria through the fusion to amitochondrial targeting sequence using Arabidopsis thaliana plants(ecotype Columbia) as a model plant. To study this we used the publishedCoxIV targeting signal (Kohler et al. Plant J 11: 613-621 (1997)) fusedto H64NORS. The construct was transformed to Arabidopsis as described inExample 10. Volatile production was determined using SPME-headspacesampling on intact Arabidopsis leaves (2-4 leaves per vial) and GC-MSanalysis using the Fisons 8060 GC-MS as described in Example 10. Nineout of twelve CoxIV-transgenic plants produced (E)-nerolidol and/ordimethylnonatriene to high levels. FIG. 31 shows representative GC-MSchromatograms for a number of transgenic lines and a wildtype.

This shows that it is possible to obtain high constitutive (andinducible) sesquiterpene biosynthesis by changing the subcellularlocation of expression of sesquiterpene genes to the mitochondria.

Example 12 Increasing the Pool of Sesquiterpene Precursors in theMitochondria by Inducing or Repressing Any of the Genes or theCorresponding Enzymatic Steps of Either the Cytosolic or the Plastidicor the Mitochondrial Isoprenoid Biosynthetic Pathway

The production of sesquiterpenes in different transgenic plant species,using their native signal sequences, has proved to be largelyunsuccesful in our hands, as has also been reported by several otherauthors (Wallaart et al., Planta 212: 460-465, 2001; Hohn and Ohlrogge,Plant Physiol 97: 460-462, 1991). We investigated how to enhance thelevel of sesquiterpene production further complementary to the methoddescribed in Example 11.

This second method depends on overexpressing an IPP isomerase or anyother gene encoding the proteins which catalyze the production ofisoprenoid precursors (i.e. DXP synthase, DXP reductoisomerase,2-C-methyl-D-erythritol 4-phosphate cytidyltransferase, 4-(cytidine 5′diphospho)-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol2,4-C-cyclodiphosphate synthase, (E)-4-hydroxy-3-methyl but-2-enyldiphosphate synthase, (E)-4-hydroxy-3-methyl but-2-enyl diphosphatereductase, acetoacetyl CoA thiolase, 3-hydroxy-3-methyl-glutaryl-CoAsynthase, 3-hydroxy-3-methyl-glutaryl-CoA reductase, MVA kinase,phosphomevanolate kinase, MVA diphosphate decarboxylase, FPP synthase)in wild-type plants or in transgenic plants, which already over-expressa sesquiterpene synthase in mitochondria using Arabidopsis thalianaplants (ecotype Columbia) as a model plant. Activity of the genes andenzymes mentioned above could either be induced or repressed to achievehigher substrate pools in the mitochondria. Since exchange of isoprenoidprecursors between different cellular compartments might occur, theabove mentioned proteins could be localized in cellular compartmentsother than mitochondria and still contribute to the overall precursorpool in mitochondria.

As an example of the above method of enhancing the pool of isoprenoidprecursors in mitochondria two genes from the above mentioned set wereintroduced in two ways, either by co-transformation of two binaryvectors harboring the different genes or by retransformation of a plantalready transformed with a single gene, and selecting using a newselectable marker (hygromycin instead of kanamycin that was used in thefirst genetic transformation). Apart from the terpenoid synthase genesdescribed elsewhere, the genes used included an IPP isomerase fromstrawberry encoding a mitochondrial protein (mitoIPPI), and a cytosolicIPP isomerase (cytoIPPI) Co-transformation was performed with thefollowing combinations of genes: mitoIPPI and a mitochondrial localizedH64NORS cytoIPPI and mitochondrial localised H64NORS

Example 13 Transgenic Plants with Improved Biological Control of Pests

Linalool and nerolidol, and its derivative4,8-dimethyl-1,3(E),7-nonatriene have been reported to play an importantrole in the attraction of predators of a variety of insect and spidermite pests by a large number of crops. The sequences described in thepresent invention can be used as markers for the selection of cropspecies, such as for example maize, cotton, apple, and cucumber, and anyother crops employing this indirect defense mechanism, with improvedproduction of volatile, predator attracting, compounds in response tofeeding herbivores. In addition, the present invention can be used tomake transgenic plants with improved signalling capacity. Hereto the DNAsequences could be placed under the control of an inducible promoter,such as wound-inducible or specific inducible promoters. These promotersare isolated from plants that were fed upon by for example spider mitesor insects. Spider mite inducible promoters can for example be isolatedfrom cucumber or lima bean. These plant species have been shown tostrongly react to spider mite feeding with the production of volatilesignalling compounds (Bouwmeester et al., 1999). Subtractive (up- anddown-regulated) libraries are made from non-infested (control) andinfested plant material using the PCR-Select™ cDNA Subtraction Kit(Clontech), and the expression of the cDNAs in these subtractivelibraries checked using cDNA micro-array technology (see for exampleAharoni et al., 2000) using mRNA from control, spider-mite infested andJA-treated plant materials as probes for hybridisation. Many inducedcDNAs are detected. The full-length cDNAs of interesting, stronglyregulated genes are obtained using the RACE PCR technology, or byscreening a cDNA library. Promoters of strongly (up-) regulated genesare isolated using the Genome Walker™ kit (Clontech).

As mentioned above, the DNA sequences from the invention can be placedunder the control of wound-inducible or the isolated suitable (tissue-)specific (inducible) promoters and used for transformation of crops inwhich biological control is enabled by the production of induciblevolatile signalling compounds, such as cucumber, maize and cotton, usingpublished protocols. As an example for the power of this approach wehave expressed the nucleic acid from the invention with a mitochondrialtargeting signal in Arabidopsis. The state of the art did not containany teaching which would predict that the expression of a cytosolicsesquiterpene synthase in mitochondria would result in high expressionof sesquiterpene compounds. Indeed, the literature which predicted thatconstitutive sesquiterpene production could be achieved by expression ofcytosolic sesquiterpene synthases in the cytosol was proved to beunreliable. Because of the presence of a mitochondrial targeted FPPsynthase in the Arabidopsis genome, we expected that FPP, the substratefor the nerolidol synthase, would be present in the mitochondria ofArabidopsis, but it did not provide any clue as to whether the substratepool would be available to sesquiterpene synthase which are normallyexpressed in the cytosol nor to which quantity the sesquiterpenes wouldbe produced.

To study this we used the published CoxIV targeting signal (Kohler etal. Plant J 11: 613-621 (1997)) fused to H64NORS. Both constructs weretransformed to Arabidopsis as described in Example 10. Volatileproduction was determined using SPME-headspace sampling on intactArabidopsis leaves (2-4 leaves per vial) and GC-MS analysis using theFisons 8060 GC-MS as described in Example 10. Nine out of twelveCoxIV-transgenic plants produced (E)-nerolidol and/or dimethylnonatrieneto very high levels, comparable to the levels of linalool, when the genewas expressed in the chloroplast. FIG. 31 shows representative GC-MSchromatograms for a number of transgenic lines and a wildtype. Bolandand cowokers have shown that a number of plant species are able toconvert exogenously applied nerolidol to dimethylnonatriene (J. Donath,W. Boland [1995] Phytochemistry 39: 785-790). Apparently, alsoArabidopsis is able to convert nerolidol to dimethylnonatriene also whennerolidol biosynthesis is catalysed by a transgene. Feeding of nerolidolto wildtype Arabidopsis leaves confirmed that nerolidol is transformedto dimethylnonatriene by endogenous Arabidopsis enzymes. The response ofpredatory mites (Phytoseiulus persimilis) to the transgenic plants wasdetermined using a Y-tube olfactometer (e.g. Takabayashi et al., J.Chem. Ecol. 20(2), 373-385, 1994). In a series of three replicatedexperiments, the dimethylnonatriene (and nerolidol) producingArabidopsis plants were highly significantly more attractive to starvedpredatory mites than wildtype Arabidopsis (P<0.001; determined using aX² test) (Table 6).

TABLE 6 Results of a two-choice Y-tube experiment in which 4 or 5CoxIV-H64NORS transformed and 4 or 5 wildtype Arabidopsis plants wereoffered to 20 predatory mites. Results were analysed using a X²-test. X²= 13.42; P < 0.001. Number of predatory mites going to Transgenic lineCoxIV-H64NORS wildtype no choice 9 18 2 8* 13 5 2 2 16 3 *According toGC-MS analysis this was a lower expressing transgenic line

Example 14 Effects of Linalool and Nerolidol Expression on Resistance toMicro-Organisms

Several plant species expressing the H64NORS gene and producing elevatedlevels of linalool and nerolidol were analyzed for resistance tomicrobial infections of powdery mildew and Phytophthora infestans. Cleareffects were observed on leaves and fruits showing that the in vitrodata presented in Example 7 are predictive of the in vivo data intransgenic plants.

Petunia and Powdery Mildew

Transformed tomato plants (control (empty vector) and transgenichomozygous for the trait) were grown from seed in a small greenhouseunder identical controlled conditions (n=30). The plants were inoculatedwith powdery mildew (Erysiphe cichoracearum) spores. After 4 weeksplants were scored for infection. The results indicate that the thepresence of linalool protected the plants from infection by mildew(Table 2).

TABLE 2 Infection of wildtype and transgenic linalool producing Petuniaplants with powdery mildew Moderately Heavily infected (lower, infectedolder leaves) Clean Control empty vector 75% — 25% Homozygous forlinalool — 10% 90%Tomato and Phytophthora infestans

Green fruits were harvested from various homozygous transgenic Microtomtomato lines. Earlier these lines had been characterized for linaloolcontent by steam destination and GC-MS. Ten different berries from eachtransgenic line were inoculated by pricking the top of the fruit with atooth pick dipped in a suspension of 10,000 sporangia/ml of Phytophthorainfestans IP0428-2. After 7 days the fruits were scored for infectionlevel (Table 3). Nearly all diseased fruits had turned completelygrey/black just below the skin. Fruits were scored clean if they had noinfection at all. A strong correlation was observed between a highlinalool expression level and a low percentage of diseased berries. Thetransgenic fruits with high linalool levels largely remained free ofinfection

TABLE 3 Relationship between linalool production and Phytophthorainfestans infection of green fruits for different transgenic lines.quantity of linalool Tomato line % diseased berries (arbitrary units)control 60 465 1A 70 2,000 1C 50 3,778 1B 30 22,989 1BA 10 18,125Potato and Phytophthora infestans

Transgenic potato lines expressing the H64NORS gene in two differentconstructs (H64NOR and H64TAR) were analyzed for production of linalooland nerolidol in the headspace using an SPME fiber and GC-MS. The H64NORconstruct did not yield nerolidol or linalool production above thebackground present in potato, while the H64TAR construct gave very highlevels of linalool and low levels nerolidol in the headspace. Both setsof plants were tested for Phytophthora infestans resistance byinoculating 5 detached leaves in 2 replicates with spore suspensions andscoring lesion area, lesion growth and sporulation (Table 4). A verystrong correlation was observed between high linalool expression levelsand strongly repressed or absent lesion growth and sporulation.

TABLE 4 Effect of different constructs on linalool production andPhytophthora infestans lesion growth and sporulation Linalool (×10²Potato ¹Lesion growth arb. units) genotypeTransgenic ¹Lesion area rate(LGR: ¹Sporulation (5 min/ Construct line (mm²) mm/day) t5-7 dpi (7 dpi)measurement) H64NOR I5 271 4.2 1.4 625 I12 506 7.7 2.8 1125 I20 294 5.22 1275 I23 456 8.0 2.6 1150 I27 574 8.5 3.4 3750 I30 458 7.0 2.3 1675H64TAR T1 0 0 0 163000 T9 79 1.6 0.8 390000 T13 123 3.7 0.8 152000 T24143 2.3 0.8 390000 T29 0 0 0 157000 T31 0 0 0 229000 ¹Five leaves pergenotype were each inoculated with 1 drop of 10 microliter inoculum perleaf (IPO428-2, 50.000 sporangia/ml) and scored for lesion area, growthand sporulation (visual score on a scale of 0-4) at the indicated dayspost infection (dpi)

FIG. 30 combines the data of table 1 and 4. FIGS. 30A, B and C providethe correlation in lesion size, lesion growth rate, and sporulationrespectively of Phytophthora infestans isolate IPO 428-2 plotted againstthe content of linalool, 8-hydroxylinalool, linalooltriol,lynalylglucoside, 8-hydroxylinalylglucoside and linalyltriolglucosidecontent of the potato transgenic lines T or TM-9, -13, -29 and a controlline. The control data from table 4 on fungal growth and sporulationwere taken to be the average values of the H64NOR plants (I-lines) withnegligible increased levels of either linalool, nerolidol orderivatives. The linalool (derivative) data provided in table 1 areknown to the art to be much more reliable and quantitative than the SPMEdata on linalool in table 4, which justifies their preferred use. FIG.30 demonstrates a strong dose-effect correlation of the levels oflinalool (derivatives) produced in potato to the levels of resistance.With high levels of terpene expression clearly complete resistance toPhytophthora infestans infection was obtained. Furthermore, FIG. 30 Dprovides the in vitro data on the sensitivity of Phytophthora infestansisolate IP0428-2 which was used for the in planta experiments to purelinalool in the medium as described in Example 9. From the comparison ofthe in vitro data with the in planta data of FIG. 30 it is clear thatthe quantities produced in planta are in the same range as thequantities required in vitro to affect the mycelial growth. It is notclear, however, whether the naturally formed alcohol and glucosidederivatives of linalool are similarly active to inhibit fungal growth asthe free underived linalool forms and may contribute to the effect offree linalool in a major way.

Example 15 Effect of Linalool and Nerolidol Expression on InsectResistance

Arabidopsis thaliana and Myzus persicae

A line of Arabidopsis thaliana transformed with the H64TAR construct wascharacterized to have a single gene insertion by Southern blot, highheadspace levels of linalool and lower headspace levels of nerolidol(Example 10). This line was selected and selfed. Selfed seeds were sownand young, non-flowering plants were analysed for levels of linalool inthe headspace using SPME GC-MS analysis (Example 10). Homo- andheterozygous plants with high levels of linalool were used in a bioassaywith Myzus persicae female adults to observe repellent or deterrenteffects of linalool expression (Table 5). For each experiment two leaveswere taken from the plant, one from a control and one from a linaloolplant and embedded next to each other in gelling wateragar of a smallpetridish. Ten adult females were placed on the inside of the lid of thepetridish. After preset times the number of adults on each of the leaveswas recorded. A deterrent effect was visible over time. Initially theaphids did not display any preference but after 2 days a verysignificant distribution was observed in which 62% were on controlplants and 38% were on linalool plants. This indicates that linalooland/or nerolidol are potential insect deterrents or repellents in plantsthat can express high levels of these compounds.

TABLE 5 Effect of linalool production in transgenic Arabidopsis onchoice of aphids. Time after Aphids on Aphids on linalool inoculationcontrol A. expressing A. Significance (hours) thaliana (%) thaliana line(%) (P-value, t-test) 0.25 50 50 0.47 1 52 47 0.31 4 58 41 0.05 20 56 430.09 24 57 42 0.2 26 59 40 0.11 45 62 37 0.003

1. An isolated nucleic acid encoding (a) the proteinaceous molecule ofSEQ ID NO: 21 or (b) a variant of the proteinaceous molecule that is atleast 95% identical to SEQ ID NO: 21 and that, under suitable reactionconditions, is capable of synthesizing at least a monoterpene alcohollinalool in the presence of geranyl diphosphate (GPP) and at least asesquiterpene alcohol nerolidol in the presence of farnesyl diphosphate(FPP).
 2. The nucleic acid according to claim 1, wherein saidproteinaceous molecule comprises nerolidol synthase/cyclase.
 3. Thenucleic acid according to claim 1, wherein the nucleic acid encodes avariant of the proteinaceous molecule that is at least 99% identical toSEQ ID NO:
 21. 4. The nucleic acid according to claim 1, which isobtainable from a eukaryote.
 5. The nucleic acid according to claim 4,wherein said eukaryote is a plant.
 6. The nucleic acid according toclaim 5, wherein said plant is a strawberry plant.
 7. The nucleic acidaccording to claim 1, which is provided with a nucleic acid encoding aplastid targeting signal.
 8. The nucleic acid according to claim 1,which is provided with a mitochondrial targeting signal.
 9. The nucleicacid according to claim 1, which encodes a proteinaceous moleculecapable of isoprenoid bioactive compound synthesis in the cytosol,plastid or mitochondrium of a cell when provided with a suitablesubstrate under appropriate reaction conditions.
 10. A vector comprisinga nucleic acid according to claim
 1. 11. A transgenic non-human hostcomprising a nucleic acid according to claim
 1. 12. The transgenic hostaccording to claim 11, wherein said host expresses a nerolidolsynthase/cyclase.
 13. The transgenic host according to claim 11, whereinsaid host is a plant or propagating material derived therefrom andcomprising said nucleic acid according to claim
 1. 14. A method forproducing a flavor, fragrance and/or bio-control agent comprising a)transforming or transfecting a suitable host with at least one nucleicacid according to claim 1; b) expressing said nucleic acid in thepresence of a suitable substrate to cause formation of product and, c)optionally, isolating the formed product.
 15. The method of claim 14,wherein said host comprises a micro-organism, plant cell or plant. 16.The method of claim 14, wherein the formed product is linalool, neridolor 4,8-dimethyl-1,3(E), 7-nonatriene.